Neurturin and related growth factors

ABSTRACT

A novel growth factor, neurturin, is disclosed. The human and mouse amino acid sequences have been identified. Human and mouse neurturin genomic DNA sequences have been cloned and sequences and the respective cDNA sequences identified. The subcloning into vectors and the preparation of cells stably transformed with the vectors is also disclosed. In addition, methods for treating degenerative conditions using neurturin, methods for detecting gene alterations and methods for detecting and monitoring patient levels of neurturin are provided. Methods for identifying additional members of the neurturin-GDNF family of growth factors are also provided.

REFERENCE TO GOVERNMENT GRANT

This invention was made with government support under Grant NumbersNS24679 and CA53524. The government has certain rights in thisinvention.

This is a divisional of copending application Ser. No. 08/519,777 filedon Aug. 28, 1999, now U.S. Pat. No. 5,739,307.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates generally to trophic or growth factors and, moreparticularly, to the novel growth factor, neurturin.

(2) Description of the Related Art

The development and maintenance of tissues in complex organisms requiresprecise control over the processes of cell proliferation,differentiation, survival and function. A major mechanism whereby theseprocesses are controlled is through the actions of polypeptides known as"growth factors" . These structurally diverse molecules act throughspecific cell surface receptors to produce these actions.

In recent years it has become apparent that growth factors fall intoclasses, i.e. families or superfamilies based upon the similarities intheir amino acid sequences. Examples of such families that have beenidentified include the fibroblast growth factor family, the neurotrophinfamily and the transforming growth factor-beta (TGF-β) family.

Of particular importance are those growth factors, termed "neurotrophicfactors", that promote the differentiation, growth and survival ofneurons and reside in the nervous system or in innervated tissues. Nervegrowth factor (NGF) was the first neurotrophic factor to be identifiedand characterized (Levi-Montalcini et al., J. Exp. Zool. 116:321, 1951which is incorporated by reference). NGF exists as a noncovalently boundhomodimer. This factor promotes the survival and growth of sympathetic,neural crest-derived sensory, and basal forebrain cholinergic neurons.In sympathetic neurons this substance produces neurite outgrowth invitro and increased axonal and dendritic growth in vivo. Earlyindications as to the physiological roles of NGF were obtained from invivo studies involving the administration of neutralizing antibodies(Levi-Montalcini and Booker, Proc Nat'l Acad Sci 46:384-391, 1960;Johnson et al. Science 210: 916-918, 1980 which are incorporated byreference), and these studies have been confirmed by analyzingtransgenic mice lacking NGF via gene targeting (Crowley et al., Cell76:1001-12, 1994 which is incorporated by reference). NGF has effects oncognition and neuronal plasticity, and can promote the survival ofneurons that have suffered damage due to a variety of mechanical,chemical, viral, and immunological insults (Snider and Johnson, AnnNeurol 26:489-506, 1989; Hefti, J Neurobiol 25:1418-35, 1994 which areincorporated by reference). NGF also is known to extensively interactwith the endocrine system and in immune and inflammatory processes.(Reviewed in Scully and Otten, Cell Biol Int 19:459-469, 1995; Otten andGadient, Int. J. Devl Neurosci 13:147-151, 1995 which are incorporatedby reference). For example, NGF promotes the survival of mast cells.(Horigome et al. J Biol Chem 269:2695-2707, 1994 which is incorporatedby reference).

It became apparent that NGF was the prototype of a family ofneurotrophic factors upon the discovery and cloning of brain-derivedneurotrophic factor (BDNF) (Liebrock et al. Nature 341:149-152, 1989which is incorporated by reference), which was the second member of thisfamily to be discovered. The relationship of BDNF to NGF, is evidencedin the conservation of all six cysteines that form the three internaldisulfides of the NGF monomer (Barde, Prog Growth Factor Res 2:237-248,1990 which is incorporated by reference). By utilizing the informationprovided by BDNF of the highly conserved portions of two factors,additional members (NT-3, NT-4/5) of this neurotrophin family wererapidly found by several groups (Klein, FASEB J 8:738-44, 1994 which isincorporated by reference). Information concerning their distributionand activities, and the physiologic consequences of their deficiencies(via gene targeting), has greatly increased our knowledge of neuronaldevelopment (for reviews, see Jelsma et al., Curr Opin Neuroblol4:717-25, 1995; Lindsay et al., Trends Neurosci 17:182-90, 1994; andJohnson et al., Curr Biol 4:662-5, 1994 which are incorporated byreference). For instance, it is now clear that the various neurotrophinsact on largely non-overlapping neuronal populations (e.g. motor neurons,sub-populations of sensory neurons), and regulate their survival andmetabolism in ways similar to those originally described for NGF. Theiridentification has also led to refinements in the neurotrophichypothesis, as evidence has accumulated that neurons can switch theirneurotrophin survival requirements during maturation (for review, seeDavies, Curr Biol 4:273-6, 1994 which is incorporated by reference).

Recently, the understanding of the mechanisms of signal transduction forneurotrophic factors has been advanced by the identification ofreceptors for the NGF family of neurotrophic factors. The tyrosinekinase receptor, trkA, identified as the NGF receptor and the closelyrelated receptors trkb, which mediates signaling of BDNF and NT-4/5, andtrkC, which mediates effects of NT-3, have allowed dissection of thesignal transduction pathways utilized by these neurotrophins (forreview, see (Tuszynski et al., Ann Neurol 35: S9-S12, 1994 which isincorporated by reference). Signaling by NGF involves proteins whichinteract directly with the phosphorylated trkA receptor (e.g. Shc,PLCγ1, PI-3 kinase), other trkA substrates like SNT (Rabin et al., MolCell Biol 13:2203-13, 1995 which is incorporated by reference), anddownstream kinase effectors (e.g. ras, rafi, MEK and MAP kinase). Insome cases, particular components have been linked to specific actionsof NGF, such as Shc and PLCγl requirement for neurite outgrowth (Loeb etal., J Biol Chem 269:8901-10, 1994; Stephens et al., Neuron 12:691-705,1994 which is incorporated by reference) and PI-3 kinase requirement forsurvival (Yao and Cooper, Science 267:2003-6, 1995 which is incorporatedby reference).

In addition to the discovery of molecules related to NGF, structurallyunrelated neurotrophic factors have also been recently identified. Theseinclude factors originally isolated based upon a "neurotrophic action"such as ciliary neurotrophic factor (CNTF) (Lin et al., Science246:1023-5, 1989 which is incorporated by reference) along with othersoriginally isolated as a result of non-neuronal activities (e.g.fibroblast growth factors (Cheng and Mattson Neuron 1:1031-41,1991 whichis incorporated by reference), IGF-I (Kanje et al, Brain Res486:396-398, 1989 which is incorporated by reference) leukemiainhibitory factor (Kotzbauer et al, Neuron 12:763-773, 1994 which isincorporated by reference).

Glial-derived neurotrophic factor (GDNF), is one such neurotrophicfactor structurally unrelated to NGF. GDNF was, thus, a unique factor,which, up until now, was not known to be a member of any subfamily offactors. The discovery, purification and cloning of GDNF resulted from asearch for factors crucial to the survival of midbrain dopaminergicneurons, which degenerate in Parkinson's disease. GDNF was purified fromrat B49 glial cell conditioned media (Lin et al., Science 260:1130-2,1993 which is incorporated by reference). Sequence analysis revealed itto be a distant member of the superfamily of transforming growth factorβ (TGF-β) factors, having approximately 20% identity based primarily onthe characteristic alignment of the 7 cysteine residues (Lin et al.,Science 260:1130-2, 1993 which is incorporated by reference). Thus, GDNFcould possibly have represented a new subfamily within the TGF-βsuperfamily.

GDNF, like other members of the TGF-β superfamily, has a precursormolecule, with a signal sequence and variably sized pro- region, that isgenerally cleaved at an RXXR site to release the 134 amino acid matureprotein, GDNF. Thus, GDNF is synthesized as a precursor protein.

Subsequent processing results in a mature glycosylated homodimer ofapproximately 35-40 kD. Six of the seven cysteines form intrachaindisulfide bonds and connect hydrogen-bonded β-sheets to make a rigidstructure called a cystine knot (McDonald et al., Cell 73:421-4, 1993which is incorporated by reference), a structure which, interestingly,is also characteristic of the neurotrophins. The remaining cysteineforms a disulfide bond with another monomer to form the biologicallyactive hetero- and homodimers. This structure may account for the strongresistance of GDNF to denaturants such as sodium dodecyl sulfate (SDS),heat and pH extremes.

Recombinant GDNF produced in bacteria specifically promotes the survivaland morphological differentiation of dopaminergic neurons in midbrainneuronal cultures (Lin et al., Science 260:1130-2, 1993 which isincorporated by reference). These initial in vitro experiments have nowbeen extended to in vivo models which demonstrate that GDNF has potentprotective and regenerative effects on MPTP- or axotomy-induced lesionsof dopaminergic neurons in adult rodent brain (Tomac et al., Nature373:335-9, 1995 and Beck et al., Nature 373:339-41, 1995 which isincorporated by reference). GDNF promotes the survival in vitro ofnodose sensory and parasympathetic neurons, and can rescue chickensympathetic neurons from NGF deprivation-induced death, but thisrequires much higher doses than are necessary for its effects ondopaminergic neurons (Ebendal et al., J Neurosci Res 40:276-84, 1995which is incorporated by reference). Significantly, GDNF is retrogradelytransported by motor neurons and is known to promote the survival ofmotor neurons inasmuch as animals treated with GDNF suffer much lessmotor neuron loss in response to lesions than untreated animals or thosetreated with other trophic factors such as CNTF, BDNF, NT-3 or NT-4/5(Henderson et al., Science 266:1062-4, 1994; Yan et al., Nature373:341-4, 1995; and Oppenheim et al., Nature 373:344-6, 1995 which areincorporated by reference). Overall, GDNF was a more potent factor forpromoting the survival of motor neurons than the other factors, and itwas the only factor that prevented neuronal atrophy in response to theselesions, thereby positioning it as a promising therapeutic agent formotor neuron diseases.

Neuronal degeneration and death occur during development, duringsenescence, and as a consequence of pathological events throughout life.It is now generally believed that neurotrophic factors regulate manyaspects of neuronal function, including survival and development infetal life, and structural integrity and plasticity in adulthood. Sinceboth acute nervous system injuries as well as chronic neurodegenerativediseases are characterized by structural damage and, possibly, bydisease-induced apoptosis, it is likely that neurotrophic factors playsome role in these afflictions. Indeed, a considerable body of evidencesuggests that neurotrophic factors may be valuable therapeutic agentsfor treatment of these neurodegenerative conditions, which are perhapsthe most socially and economically destructive diseases now afflictingour society. Nevertheless, because different neurotrophic factors canact preferentially through different receptors and on different neuronalcell types, there remains a continuing need for the identification ofnew members of neurotrophic factor families for use in the diagnosis andtreatment of a variety of acute and chronic diseases of the nervoussystem.

SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to theidentification and isolation of substantially purified factors thatpromote the survival and growth of neurons. Accordingly, the inventorsherein have succeeded in discovering a novel protein growth factorreferenced herein as neurturin. This growth factor is believed to showat least 85% sequence identity among homologous sequences from differentmammalian species although sequence homology may be as low as 65% innon-mammalian species such as avian species. Neurturin proteinsidentified herein include the human sequence as set forth in SEQ ID NO:1(FIG. 5; FIG. 7, amino acid residues 96 through 197) and the mousesequence as set forth in SEQ ID NO:2 (FIG. 5; FIG. 8, amino acidresidues 96 through 195).

Neurturin has been identified and obtained from conditioned medium ofthe Chinese hamster ovary cells, DG44CHO-pHSP-NGFI-B cells, hereinafterreferenced as CHO cells and the factor as isolated from these cells hasan apparent molecular weight of approximately 20-30 kD as determined bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)under non-reducing conditions and an EC₅₀ in a superior cervicalganglion survival assay of less than about 10 ng/ml. The proteinisolated from Chinese hamster ovary cells is believed to be ahomodimeric protein whose monomers have an apparent molecular weight ofapproximately 10-15 kD.

Neurturin, can also be identified on the basis of fragments obtainedfollowing partial digestion of the factor isolated from CHO cellconditioned medium wherein some of the amino acid residues were not atthe time known with certainty. Such fragments include an N-terminalfragment,Ser-Gly-Ala-Arg-Pro-Xaa-Gly-Leu-Arg-Glu-Leu-Glu-Val-Ser-Val-Ser whereXaa was an unknown amino acid (SEQ ID NO:3) and internal amino acidfragments, Xaa₁ -Cys-Ala-Gly-Ala-Xaa₂ -Glu-Ala-Ala-Val where Xaa₁ wasunknown amino acid, Xaa₂ was Ser or Cys (SEQ ID NO:4), Xaa₁ -Xaa₂-Val-Glu-Ala-Lys-Pro-Cys-Cys-Gly-Pro-Thr-Ala-Tyr-Glu-Asp-Xaa₃-Val-Ser-Phe-Leu-Ser-Val where Xaa₁ and Xaa₂ were unknown, Xaa₃ was Glnor Glu (SEQ ID NO:5) and Tyr-His-Thr-Leu-Gln-Glu-Leu-Ser-Ala-Arg (SEQ IDNO:6).

A pre-pro form of neurturin is cleaved to form the mature protein andthe human pre-pro form containing the pre-pro region and the matureneurturin sequence for human is as set forth in SEQ ID NO:7 (FIG. 7,amino acid residues 1 through 197). The mouse pre-pro form is as setforth in SEQ ID NO:8 (FIG. 8, amino acid residues 1 through 195).

The present invention also provides nucleotide sequences that encode thehuman neurturin as set forth in the amino acid sequence of SEQ ID NO:1and the mouse neurturin as set forth in the amino acid sequence of SEQID NO:2. The human sequence is further identified as being encoded bythe nucleotide sequence of SEQ ID NO:9 (FIG. 7, nucleic acid 286 throughnucleic acid 591) and the mouse sequence is further identified as beingencoded by the nucleotide sequence of SEQ ID NO:10 (FIG. 8, nucleic acid286 through nucleic acid 585). Also provided are the nucleotidesequences that encode the human pre-pro neurturin as set forth in theamino acid sequence of SEQ ID NO:7 and the mouse pre-pro neurturin asset forth in the amino acid sequence of SEQ ID NO:8. The human pre-proneurturin sequence is further identified as being encoded by thenucleotide sequence of SEQ ID NO:11 (FIG. 7, nucleic acid 1 throughnucleic acid 591) and the mouse pre-pro neurturin sequence is furtheridentified as being encoded by the nucleotide sequence of SEQ ID NO:12(FIG. 8, nucleic acid 1 through nucleic acid 585).

Expression vectors and stably transformed cells are also provided. Thetransformed cells can be used in a method for producing neurturin.

In another embodiment, the present invention provides a method forpreventing or treating neuronal degeneration comprising administering toa patient in need thereof a therapeutically effective amount ofneurturin. A patient may also be treated by implanting transformed cellswhich express neurturin or a DNA sequence which encodes neurturin into apatient, or cells cultured and expanded by growth in neurturin.

The present invention also provides compositions and methods fordetecting neurturin. One method is based upon neurturin antibodies andother methods are based upon detecting neurturin mRNA using recombinantDNA techniques.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of a new growth factor,neurturin, which can maintain and prevent the atrophy, degeneration ordeath of certain cells, in particular neurons; the provision of othermembers of the neurturin-GDNF family of growth factors by makingavailable new methods capable of obtaining said other family members;the provision of methods for obtaining neurturin by recombinanttechniques and by isolation from cells; the provision of methods forpreventing or treating diseases producing cellular degeneration and,particularly neuronal degeneration; the provision of methods that candetect and monitor neurturin levels in a patient; and the provision ofmethods that can detect alterations in the neurturin gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the purification scheme for preparing neurturin fromCHO cells;

FIG. 2 illustrates the characterization of fractions eluted from Mono Scolumn in purifying neurturin showing (a) electrophoresis of eachfraction on a SDS-polyacrylamide gel and visualization of the proteinsby silver stain and (b) the neurotrophic activity present in eachfraction in the superior cervical ganglion survival assay;

FIG. 3A-3C illustrate the ability of neurturin to maintain survival ofsuperior cervical ganglionic cells in culture showing (a) positivecontrol cells maintained with nerve growth factor (NGF) (b) negativecontrol cells treated with anti-NGF antibodies showing diminishedsurvival and (c) cells treated with anti-NGF and neurturin(approximately 3 ng/ml) showing survival of neurons;

FIG. 4 illustrates the concentration-response effect of neurturin in thesuperior cervical ganglion survival assay;

FIG. 5 illustrates the homology of the amino acid sequences for themature growth factors, human neurturin (hNTN), mouse neurturin (mNTN),rat GDNF (rGDNF), mouse GDNF (mGDNF) and human GDNF (hGDNF) withidentical amino acid residues enclosed in boxes;

FIG. 6 illustrates the tissue distribution of neurturin mRNA and themRNA for GDNF using RT/PCR analysis on RNA samples obtained fromembryonic day 21 (E21) and adult rats;

FIG. 7 illustrates the cDNA and encoded amino acid sequence of humanpre-pro neurturin (SEQ ID NO:11) showing the pre- region from nucleicacid 1 through 57 (SEQ ID NO:17), the pro- region from nucleic acid 58through 285 (SEQ ID NO:20), human neurturin from nucleic acid 286through 591 (SEQ ID NO:9) and the splice site between nucleic acids 169and 170 which defines the coding sequence portion of two exons fromnucleic acids 1 through 169 (SEQ ID NO:27) and 170 through 594 (SEQ IDNO:28);

FIG. 8 illustrates the cDNA and encoded amino acid sequence of mousepre-pro neurturin (SEQ ID NO:12) showing the pre- region from nucleicacid 1 through 57 (SEQ ID NO:18), the pro- region from nucleic acid 58through 285 (SEQ ID NO:21), mouse neurturin from nucleic acid 286through 585 (SEQ ID NO:10) and the splice site between nucleic acids 169and 170 which defines the coding sequence portion of two exons fromnucleic acids 1 through 169 (SEQ ID NO:29) and 170 through 588 (SEQ IDNO:30);

FIG. 9 illustrates the mouse cDNA sequence containing a 5' non-codingregion (SEQ ID NO:13) and a 3' non-coding region (SEQ ID NO:14) each ofwhich are contiguous to the coding region of pre-pro neurturin; and

FIG. 10 illustrates the percent neuronal survival in E18 rat nodoseganglia neurons treated 24 hours post-plating for NTN, GDNF, BDNF, NGFand AMO.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based upon the identification, isolation andsequencing of a new growth factor, neurturin. Surprisingly, thissubstance has been discovered to be able to promote cell survival and,in particular, the survival of neurons. Prior to this invention,neurturin was unknown and had not been identified as a discretebiologically active substance nor had it been isolated in pure form.

The inventors herein have succeeded in discovering and isolatingneurturin from conditioned medium for CHO cells. The initial neuronalsurvival promoting activity was identified by the inventors in apartially purified preparation of this CHO-conditioned medium.Preparation of conditioned medium for a given cell line is well known inthe art (for example, see Reid, in Methods in Enzymology Vol. LVIII,Cell Culture, Jakoby and Pastan, Eds., Academic Press, San Diego, pp161-164, 1979; Freshney, Culture of Animal Cells in A Manual of BasicTechnique, 2d Ed., Wiley-Liss, N.Y., p. 84, 1987 which are incorporatedby reference). Thus, although in the present work CHO cells werecultured and the conditioned medium used to identify and to obtainneurturin in purified form, one skilled in the art will readilyappreciate that any cell that expresses neurturin can be used as asource. Some of the cells that express neurturin are identified below inExample 9 and the inventors herein believe that any of the cellsidentified as expressing neurturin can be used to obtain conditionedmedium from which neurturin can be isolated.

In the isolation of neurturin from the CHO cell conditioned medium, aninitial crude conditioned medium can be obtained by centrifugationand/or filtration to remove cellular debris. For further purification,one skilled in the art will readily appreciate that any of a number ofmethods known in the art can be used to isolate and purify neurturinfrom a biological sample such as affinity chromatography, ion exchangechromatography, preparative electrophoresis or the like wherein themethods are used either individually or in combination.

The cell survival promoting effect of neurturin can be assessed in anysuitable system for assessing cell survival. The inventors-hereinbelieve that neurturin can promote survival in a variety of differenttissues based upon what is known for other growth factors and upon theobservation that neurturin is expressed in a number of tissues in whichit is believed to have a survival promoting effect. In the work reportedherein, neuronal activity was assessed using a sympathetic neuronalsurvival assay (sympathetic cervical ganglia, SCG) which has beenextensively characterized (Martin et al, J Cell Biol 106:829-844, 1989;Deckwerth and Johnson, J Cell Biol 123:1207-1222, 1993 which areincorporated by reference) (see FIG. 3). We also show the survivalpromoting effects of neurturin on sensory neurons (See FIG. 10).

The SCG assay involved, in brief, the culturing of cells obtained fromsuperior cervical ganglia of rat embryo for 5 days at 37° C. in mediumcontaining nerve growth factor (NGF). The medium was then exchanged witha medium containing no NGF and containing anti-NGF antiserum. Removal ofNGF results normally in death of the neurons in 24-72-hours. Neuronalsurvival was visually assessed under a microscope on days 7-8. Maximumneuronal survival criteria included lack of degeneration of bothneuronal cell bodies and neurites. Cell body degeneration was indicatedwhen the neuronal cell body was reduced in size, showed irregularmembrane swellings, contained vacuoles, or had lost refractility. Afield of neurites was scored as showing signs of disintegration whenswellings and blebs appeared along the neurite bundles. Survival wasdetermined by comparison with neurons grown in the presence of NGF(positive control) or in the absence of NGF with NGF antisera (negativecontrol).

Activity was quantitated by calculation of a "survival unit". The totalsurvival units in a sample were defined as the minimal volume of analiquot of the sample which produced maximal survival divided into thetotal volume of that sample. For example, a volume of 600 ml was elutedfrom the heparin agarose column and from this eluate, 12.5 μl was theminimum volume that promoted maximal volume. Thus, the survival units inthe eluate from the heparin agarose column was 48,000. Specific activitywas calculated as the survival units divided by the mg total protein.The intrinsic activity of neurturin is expressed herein in concentrationunits of pg/ml or pM promoting maximal or half-maximal survival. Asshown in FIG. 5, a concentration-response curve of purified neurturinprotein indicates that the intrinsic activity of neurturin expressed asan EC₅₀ is approximately 1.5 ng/ml or approximately 50 pM and an EC₁₀₀is approximately 3 ng/ml or approximately 100 pM.

Survival units were determined in an assay using approximately 1200neurons in a 0.5 ml culture assay and a culture period of 48 hoursfollowing addition of the fraction. Survival was assessed visually afterthe 48 hours. Intrinsic activity as shown in FIG. 4 was determined in anassay using approximately 2700 neurons and a culture period of 72 hours.Survival was assessed by fixing the neurons and counting the number ofsurviving neurons. Because the stability, as assessed by half-life ofactivity, for neurturin decreases as the number of neurons increases,the intrinsic activity measurement would be expected to be lower thanthat predicted by Specific Activity determinations. The intrinsicactivity measurement would also be expected to be lower than thatpredicted by specific activity because the survival was measured after72 hours instead of 48 hours.

The purification of neurturin is described in detail in Example 1 below.The conditioned medium starting material was prepared from a derivativeof DG44 Chinese hamster ovary cells, DG44CHO-pHSP-NGFI-B (Day et al, JBiol Chem 265:15253-15260, 1990 which is incorporated by reference). Theinventors herein have also isolated neurturin in partially purified formfrom conditioned medium of other derivatives of DG44 Chinese hamsterovary cells and these other cells could be used equally as well as theDG44CHO-pHSP-NGFI-B cells as could the parent DG44 Chinese hamster ovaryCells, ovary cells from other species and cells from other tissues suchas those known to express neurturin (See example 9). In preparing theconditioned medium, cells were placed in serum free medium for 2 days atwhich time conditioned medium is collected and the medium replenished.This cycle was repeated to yield 5 harvests of conditioned medium fromeach batch of CHO cells. The collected media was centrifuged to removecellular debris.

The first step in purification of neurturin from the CHO cellconditioned medium involved the introduction of the conditioned mediumonto a heparin agarose column and the elution of partially purifiedneurturin therefrom. This step resulted in an 111 fold increase in thespecific activity and purification of the protein. The buffer used toapply the medium to the column contains 0.5M NaCl. At this concentrationof NaCl the neurturin binds to the heparin agarose matrix. The inventorsherein believe that based upon their isoelectric points, LIF and CNTFwould either not bind to the heparin agarose matrix or be washed awayfrom the matrix with buffer containing 0.5M NaCl. Thus, this step wouldbe expected to isolate neurturin from growth factors such as LIF andCNTF. After washing the column, neurturin was eluted from the columnusing 1.0M NaCl.

For further purification, the eluted material was then diluted andintroduced into a column containing SP SEPHAROSE® High Performance ionexchange resin (Pharmacia, Piscataway, N.J.). Material eluted from thiscolumn was further purified using fast protein liquid chromatography(FPLC) on a Chelating Superose HR 10/2 column charged with Cu⁺⁺(Pharmacia, Piscataway, N.J.). Eluted fractions from the Cu⁺⁺ superosecolumn were introduced into a Mono S HR 5/5 cation exchange column(Pharmacia, Piscataway, N.J.) for further FPLC purification. Thecomposition of the proteins in the Mono S fractions were analyzed usingnon-reducing SDS-PAGE and silver staining.

Fractions collected from the columns at each stage of purification wereassayed for biological activity using the neuronal survival assay andfor protein content using the dye binding method of Bradford (AnalBiochem 72:248-254, 1976 which is incorporated by reference) with aBio-Rad protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules,Calif.). The progressive purification using the above steps is shown intable 1.

                  TABLE 1                                                         ______________________________________                                                              Specific                                                       Protein.sup.a                                                                       Activity.sup.b                                                                         Activity.sup.d                                                                         Yield Purification                                    (mg)  (units)  (units/mg)                                                                             (%)   (fold)                                   ______________________________________                                        Conditioned                                                                            5000    48000.sup.c                                                                            9.6    --    --                                     Medium                                                                        Heparin  45      48000    1068   100   111                                    Agarose                                                                       SP Sepharose                                                                           5.3     48000    9058   100   943                                    Cu++ Superose                                                                          0.31    30000    96700  62    10070                                  Mono S   0.004   15000    3750000                                                                              31    390000                                 ______________________________________                                         .sup.a mg protein was determined using the dye binding method of Bradford     (Anal Biochem 72:248, 1976).                                                  .sup.b The total activity units or survival units in a sample were define     as the minimal volume of an aliquot of the sample which produced maximal      survival divided into the total volume of that sample.                        .sup.c Activity for Conditioned Medium was derived from the assumption        that 100% of the activity was recovered in the heparin agarose fraction       because the activity of conditioned medium was too low to be directly         assayed.                                                                      .sup.d Specific Activity was the Activity units divided by the mg total       protein.                                                                 

The results of this analysis along with the results of the neuronalsurvival assay of fractions revealed that a protein having an apparentmolecular weight of about 25 kD co-purified with the sympathetic neuronsurvival activity.

The purified material isolated from CHO cell conditioned medium was usedto determine partial amino acid sequences of the protein in CHO cellconditioned medium and subsequently as a basis for determining thesequences in different species. The N-terminal amino acid sequence wasdetermined using an automated protein/peptide sequencer and the first 16amino acids were considered to be, with uncertainty as to position 6,Ser-Gly-Ala-Arg-Pro-Xaa-Gly-Leu-Arg-Glu-Leu-Glu-Val-Ser-Val-Ser whereXaa was an unknown amino acid (SEQ ID NO:3). Internal amino acidfragments were obtained from the purified material following digestionwith protease enzymes and the sequences determined. Three internalfragments thus obtained were (1) with uncertainty as to positions 1, 2and 6, Xaa₁ -Cys-Ala-Gly-Ala-Xaa₂ -Glu-Ala-Ala-Val where Xaa₁ wasunknown amino acid, Xaa₂ was Ser or Cys (SEQ ID NO:4); (2) withuncertainty as to positions 1, 2, 4, 10, 17 and 22, Xaa₁ -Xaa₂-Val-Glu:-Ala-Lys-Pro-Cys-Cys-Gly-Pro-Thr-Ala-Tyr-Glu-Asp-Xaa₃-Val-Ser-Phe-Leu-Ser-Val where Xaa₁ and Xaa₂ were unknown, Xaa₃ was Glnor Glu (SEQ ID NO:5) and (3) Tyr-His-Thr-Leu-Gln-Glu-Leu-Ser-Ala-Arg(SEQ ID NO:6). Based upon these partial amino acid sequences, DNA probesand primers can be made and used to obtain cDNA clones from differentspecies based upon high sequence conservation between mammalian species.The human cDNA and inferred amino acid sequence is shown in FIG. 7 andthe mouse cDNA and inferred amino acid sequence is shown in FIG. 8.

The cDNA clone from mouse was 1.0 kb having an open reading frame of 585nucleotides (SEQ ID NO:12) encoding the mouse pre-pro neurturin protein(SEQ ID NO:8, FIG. 8). In addition, non-coding regions have beenidentified at both the 5' and 3' ends of the coding region as shown inFIG. 9. (SEQ ID NO:13, 5' non-coding region, nucleic acids -348 through-1; SEQ ID NO:14, 3' non-coding region, nucleic acids 589 through 675).The mouse neurturin sequence can be used to obtain PCR primers for usein identifying homologs from other species. A human 192 nucleotidefragment from human genomic DNA was amplified by this method and furtherused to screen a human genomic library to obtain clones containing thehuman neurturin genomic locus. The human cDNA sequence was deduced fromthe sequencing of these clones. (FIG. 7, cDNA sequence of human pre-proneurturin).

Reference to neurturin herein is intended to be construed to includegrowth factors of any origin which are substantially homologous to andwhich are biologically equivalent to the neurturin characterized anddescribed herein. Such substantially homologous growth factors may benative to any tissue or species and, similarly, biological activity canbe characterized in any of a number of biological assay systems.Reference to pre-pro neurturin herein is intended to be construed toinclude pre-pro growth factors containing a pre- or leader or signalsequence region, a pro- sequence region and neurturin as defined herein.

The terms "biologically equivalent" are intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same growth properties in a similar fashion, notnecessarily to the same degree as the neurturin isolated from the CHOcell conditioned medium herein or recombinantly produced human or mouseneurturin.

By "substantially homologous" it is meant that the degree of homology ofhuman and mouse neurturin to neurturin from any species is greater thanthat between neurturin and any previously reported member of the TGF-βsuperfamily or GDNF (For discussion of homology of TGF-β superfamilymembers see Kingsley, Genes and Dev 8:133-46, 1994 which is incorporatedby reference).

Sequence identity or percent identity is intended to mean the percentageof same residues between two sequences, referenced to human neurturinwhen determining percent identity with non-human neurturin, referencedto neurturin when determining percent identity with non-neurturin growthfactors and referenced to human GDNF when determining percent identityof non-neurturin growth factors with GDNF, when the two sequences arealigned using the Clustlal method (Higgins et al, Cabios 8:189-191,1992) of multiple sequence alignment in the Lasergene biocomputingsoftware (DNASTAR, INC, Madison, Wis.). In this method, multiplealignments are carried out in a progressive manner, in which larger andlarger alignment groups are assembled using similarity scores calculatedfrom a series of pairwise alignments. Optimal sequence alignments areobtained by finding the maximum alignment score, which is the average ofall scores between the separate residues in the alignment, determinedfrom a residue weight table representing the probability of a givenamino acid change occurring in two related proteins over a givenevolutionary interval. Penalties for opening and lengthening gaps in thealignment contribute to the score. The default parameters used with thisprogram are as follows: gap penalty for multiple alignment=10; gaplength penalty for multiple alignment=10; k-tuple value in pairwisealignment=1; gap penalty in pairwise alignment=3; window value inpairwise alignment=5; diagonals saved in pairwise alignment=5. Theresidue weight table used for the alignment program is PAM250 (Dayhoffet al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NDRF,Washington, Vol. 5, suppl. 3, p. 345, 1978).

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to human neurturinwhen determining percent conservation with non-human neurturin,referenced to neurturin when determining percent conservation withnon-neurturin growth factors, and referenced to human GDNF whendetermining percent conservation to non-neurturin growth factors withGDNF. Conservative amino acid changes satisfying this requirement are:R-K; E-D, Y-F, L-M; V-I, Q-H. The calculations of identity (I) andconservation (C) between mature human and mature mouse neurturin (hNTNand mNTN, respectively) and between each of these and mature human, ratand mouse GDNF (hGDNF, rGDNF and mGDNF, respectively) are shown in table2.

                  TABLE 2                                                         ______________________________________                                        COMPARISON   % IDENTITY % CONSERVATION                                        ______________________________________                                        hNTN v. mNTN 90         93                                                    hNTN v. rGDNF                                                                              44         53                                                    hNTN v. mGDNF                                                                              43         52                                                    hNTN v. hGDNF                                                                              43         53                                                    mNTN v. rGDNF                                                                              42         52                                                    mNTN v. mGDNF                                                                              41         51                                                    mNTN v. hGDNF                                                                              41         52                                                    ______________________________________                                    

The degree of homology between the mature mouse and human neurturinproteins is about 90% sequence identity and all neurturin homologs ofnon-human mammalian species are believed to similarly have at leastabout 85% sequence identity with human neurturin. For non-mammalianspecies such as avian species, it is believed that the degree ofhomology with neurturin is at least about 65% identity. By way ofcomparison, the variations between family members of the neurturin-GDNFfamily of growth factors can be seen by comparing neurturin and GDNF.Human and mouse neurturin have about 40% sequence identity and about 50%sequence conservation with human, mouse and rat GDNF. It is believedthat the different family members similarly have a sequence identity ofabout 40% of that of neurturin and about 40% of that of GDNF and withina range of about 30% to about 85% identity with neurturin and within arange of about 30% to about 85% sequence identity with GDNF. Thus, agiven non-neurturin and non-GDNF family member from one species would beexpected to show lesser sequence identity with neurturin and with GDNFfrom the same species than the sequence identity between human neurturinand neurturin from a non-human mammalian species, but greater sequenceidentity than that between human neurturin and any other known member ofthe TGF-β superfamily member except GDNF (Kingsley, supra). In the caseof pre-pro neurturin, homologs of pre-pro neurturin in non-humanmammalian species can be identified by virtue of the neurturin portionof the amino acid sequence having at least about 85% sequence identitywith human neurturin and homologs of pre-pro neurturin in non-mammalianspecies can be identified by virtue of the neurturin portion of theamino acid sequence having at least about 65% identity with humanneurturin.

Neurturin can also include hybrid and modified forms of neurturinincluding fusion proteins and neurturin fragments and hybrid andmodified forms in which certain amino acids have been deleted orreplaced and modifications such as where one or more amino acids havebeen changed to a modified amino acid or unusual amino acid andmodifications such as glycosolations so long as the hybrid or modifiedform retains the biological activity of neurturin. By retaining thebiological activity, it is meant that neuronal survival is promoted,although not necessarily at the same level of potency as that of theneurturin isolated from CHO cell conditioned medium or that of therecombinantly produced human or mouse neurturin.

Also included within the meaning of substantially homologous is anyneurturin which may be isolated by virtue of cross-reactivity withantibodies to the neurturin described herein or whose encodingnucleotide sequences including genomic DNA, mRNA or cDNA may be isolatedthrough hybridization with the complementary sequence of genomic orsubgenomic nucleotide sequences or cDNA of the neurturin herein orfragments thereof. It will also be appreciated by one skilled in the artthat degenerate DNA sequences can encode human neurturin and these arealso intended to be included within the present invention as are allelicvariants of neurturin.

In the case of pre-pro neurturin, alternatively spliced protein productsresulting from an intron located in the coding sequence of the proregion may exist. The intron is believed to exist in the genomicsequence at a position corresponding to that between nucleic acids 169and 170 of the cDNA which, in turn, corresponds to a position withinamino acid 57 in both the mouse and human pre-pro neurturin sequences(see FIGS. 7 and 8). Thus, alternative splicing at this position mightproduce a sequence that differs from that identified herein for humanand mouse pre-pro neurturin (SEQ ID NO:11 and SEQ ID NO:12,respectively) at the identified amino acid site by addition and/ordeletion of one or more amino acids. Any and all alternatively splicedpre-pro neurturin proteins are intended to be included within the termspre-pro neurturin as used herein.

Although it is not intended that the inventors herein be bound by anytheory, it is thought that the human and mouse proteins identifiedherein as well as homologs from other tissues and species may exist asdimers in their biologically active form in a manner consistent withwhat is known for other factors of the TGF-β superfamily.

In addition to homodimers, the monomeric units of the dimers ofneurturin can be used to construct stable growth factor heterodimers orheteromultimers comprising at least one monomer unit derived fromneurturin. This can be done by dissociating a homodimer of neurturininto its component monomeric units and reassociating in the presence ofa monomeric unit of a second homodimeric growth factor. This secondhomodimeric growth factor can be selected from a variety of growthfactors including GDNF or a member of the NGF family such as NGF, BDNF,NT-3 and NT-4/5 or a member of the TGF-β superfamily, or a vascularendothelial growth factor or a member of the CNTF/LIF family or thelike.

Growth factors are thought to act at specific receptors. For example,the receptors for TGF-β and activins have been identified and make up afamily of Ser/Thr kinase transmembrane proteins (Kingsley, Genes and Dev8:133-146, 1994; Bexk et al Nature 373:339-341, 1995 which areincorporated by reference). In the NGF family, NGF binds to the TrkAreceptor in peripheral sensory and sympathetic neurons and in basalforebrain neurons; BDNF and NT-4/5 bind to trkB receptors; and NT-3binds primarily to trkC receptors that possess a distinct distributionwithin the CNS (Tuszynski et al., Ann Neurol 35:S9-S12, 1994). Theinventors herein believe that GDNF, neurturin and as yet unknown membersof this family of growth factors act through specific receptors havingdistinct distributions as has been shown for other growth factorfamilies. Thus, by forming heterodimers or heteromultimers of neurturinand one or more other growth factors, the resultant growth factor wouldbe expected to be able to bind to at least two distinct receptor typespreferentially having a different tissue distribution. The resultantheterodimers or heteromultimers would be expected to show an enlargedspectrum of cells upon which it could act or provide greater potency. Itis also possible that the heterodimer or heteromultimer might providesynergistic effects not seen with homodimers or homomultimers. Forexample, the combination of factors from different classes has beenshown to promote longterm survival of oligodendrocytes whereas singlefactors or combinations of factors within the same class promotedshort-term survival (Barres et al., Development 118:283-295, 1993).

Heterodimers can be formed by a number of methods. For example,homodimers can be mixed and subjected to conditions in whichdissociation/unfolding occurs, such as in the presence of adissociation/unfolding agent, followed by subjection to conditions whichallow monomer reassociation and formation of heterodimers.Dissociation/unfolding agents include any agent known to promote thedissociation of proteins. Such agents include, but are not limited to,guanidine hydrochloride, urea, potassium thiocyanate, pH lowering agentssuch as buffered HCl solutions, and polar, water miscible organicsolvents such as acetonitrile or alcohols such as propanol orisopropanol. In addition, for homodimers linked covalently by disulfidebonds as is the case with TGF-β family members, reducing agents such asdithiothreitol and β-mercaptoethanol can be used fordissociation/unfolding and for reassociation/refolding.

Heterodimers can also be made by transfecting a cell with two or morefactors such that the transformed cell produces heterodimers as has beendone with neurotrophin. (Heymach and Schooter, J Biol Chem270:12297-12304, 1995).

Another method of forming heterodimers is by combining neurturinhomodimers and a homodimer from a second growth factor and incubatingthe mixture at 37° C.

When heterodimers are produced from homodimers, the heterodimers maythen be separated from homodimers using methods available to thoseskilled in the art such as, for example, by elution from preparative,non-denaturing polyacrylamide gels. Alternatively, heterodimers may bepurified using high pressure cation exchange chromatography such as witha Mono S cation exchange column or by sequential immunoaffinity columns.

It is well known in the art that many proteins are synthesized within acell with a signal sequence at the N-terminus of the mature proteinsequence and the protein carrying such a leader sequence is referred toas a preprotein. The pre- portion of the protein is cleaved duringcellular processing of the protein. In addition to a pre- leadersequence, many proteins contain a distinct pro sequence that describes aregion on a protein that is a stable precursor of the mature protein.Proteins synthesized with both pre- and pro- regions are referred to aspreproproteins. In view of the processing events known to occur withother TGF-β family members as well as the sequences determined herein,the inventors believe that the form of neurturin protein as synthesizedwithin a cell is the pre-pro neurturin. The pre-pro neurturin isbelieved to contain an N-terminal 19 amino acid signal sequence (humanpre- signal sequence, SEQ ID NO:15, FIG. 7, amino acids 1 through 19encoded by SEQ ID NO:17, FIG. 7, nucleic acids 1 through 57; mouse pre-signal sequence, SEQ ID NO:16, FIG. 8, amino acids 1 through 19, encodedby SEQ ID NO:18, FIG. 8, nucleic acids 1 through 57). It is known thatthe full length of a leader sequence is not necessarily required for thesequence to act as a signal sequence and, therefore, within thedefinition of pre- region of neurturin is included fragments thereof,usually N-terminal fragments, that retain the property of being able toact as a signal sequence, that is to facilitate co-translationalinsertion into the membranes of one or more cellular organelles such asendoplasmic reticulum, mitochondria, golgi, plasma membrane and thelike.

The signal sequence is followed by a pro-domain which contains an RXXRproteolytic processing site immediately before the N-terminal amino acidsequence for the mature neurturin. (human pro- region sequence, SEQ IDNO:19, FIG. 7, amino acids 20 through 95 encoded by the nucleic acidsequence SEQ ID NO:20, FIG. 7 nucleic acids 58 through 285; mouse pro-region sequence, SEQ ID NO:22, FIG. 8, amino acids 19 through 95 encodedby nucleic acid sequence SEQ ID NO:21, FIG. 8, nucleic acids 58 through285).

The pre- and pro- regions together comprise a pre-pro sequenceidentified as the human pre-pro sequence (SEQ ID NO:23, FIG. 7, aminoacids 1 through 95 encoded by SEQ ID NO:25, nucleic acids 1 through 285)and the mouse pre-pro sequence (SEQ ID NO:24, FIG. 8, amino acids 1through 95 encoded by SEQ ID NO:26, nucleic acids 1 through 285). Thepre- region sequences and pro- region sequences as well as the pre-proregion sequences can be identified and obtained for non-human mammalianspecies and for non-mammalian species by virtue of the sequences beingcontained within the pre-pro neurturin as defined herein.

Using the above landmarks, the mature, secreted neurturin molecule ispredicted to be approximately 11.5 kD which is likely to form adisulfide linked homodimer of approximately 23 kD by analogy to othermembers of the TGF-β family. The predicted approximately 23 kD proteinis consistent with the 25 kD protein purified from CHO cell conditionedmedia being a homodimer. The inventors herein have detected anapproximately 11.5 kD protein from conditioned medium of Chinese hamsterovary cells transfected with the neurturin expression vector(pCMV-NTN-3-1) using SDS-PAGE under reducing conditions and this proteinis thought to be the monomer.

The nucleotide sequences of pre- and/or pro- regions can also be used toconstruct chimeric genes with the coding sequences of other growthfactors or proteins and, similarly, chimeric genes can be constructedfrom the coding sequence of neurturin coupled to sequences encoding pre-and/or pro- regions from genes for other growth factors or proteins.(Booth et al., Gene 146:303-8, 1994; Ibanez, Gene 146:303-8, 1994;Storici et al., FEBS Letters 337:303-7, 1994; Sha et al J Cell Biol114:827-839, 1991 which are incorporated by reference). Such chimericproteins can exhibit altered production or expression of the activeprotein species.

A preferred neurturin of the present invention has been identified andisolated in purified form from medium conditioned by CHO cells. Alsopreferred is neurturin prepared by recombinant DNA technology. By "pureform" or "purified form" or "substantially purified form" it is meantthat a neurturin composition is substantially free of other proteinswhich are not neurturin.

Recombinant human neurturin may be made by expressing the DNA sequencesencoding neurturin id a suitable transformed host cell. Using methodswell known in the art, the DNA encoding neurturin may be linked to anexpression vector, transformed into a host cell and conditionsestablished that are suitable for expression of neurturin by thetransformed cell.

Any suitable expression vector may be employed to produce recombinanthuman neurturin such as, for example, the mammalian expression vectorpCB6 (Brewer, Meth Cell Biol 43:233-245, 1994) or the E. coli pETexpression vectors, specifically, pET-30a (Studier et al., MethodsEnzymol 185:60-89, 1990 which is incorporated by reference) both ofwhich were used herein. Other suitable expression vectors for expressionin mammalian and bacterial cells are known in the art as are expressionvectors for use in yeast or insect cells. Baculovirus expression systemscan also be employed.

Neurturin may be expressed in the monomeric units or such monomeric formmay be produced by preparation under reducing conditions. In suchinstances refolding and renaturation can be accomplished using one ofthe agents noted above that is known to promote dissociation/associationof proteins. For example, the monomeric form can be incubated withdithiothreitol followed by incubation with oxidized glutathione disodiumsalt followed by incubation with a buffer containing a refolding agentsuch as urea.

By analogy with the N-terminal sequence and internal fragments of theneurturin purified from CHO cell conditioned medium, the mature mousesequence was deduced and from this the mature human form was predictedusing the sequence from the human gene. The amino acid sequence of themature human form is as shown in FIG. 5 (hNTN, SEQ ID NO:1). Thematerial purified from CHO cell conditioned medium is considered to bemature neurturin and may exist as a dimer or other multimer and may beglycosylated or chemically modified in other ways. As noted above, themouse and human nucleic acid sequences suggest that neurturin isinitially translated as a pre-pro polypeptide and that proteolyticprocessing of the signal sequence and the "pro" portion of this moleculeresults in the mature sequence, referenced herein as "mature neurturin",as obtained from medium condition by CHO cells and as exists in humanand in non-human species in homologous form. The present invention,therefore, includes any and all "mature neurturin" sequences from humanand non-human species and any and all pre-pro neurturin polypeptidesthat may be translated from the neurturin gene.

It is believed that the coding sequence for the pre-pro-neurturinpolypeptide begins at the first ATG codon encoding methionine at the 5'end of the clone (position 1 in FIG. 9) which is positioned in the samereading frame as the sequence encoding the amino acid sequences obtainedfrom the purified neurturin. Downstream from the first codon is thelargest open reading frame containing the coding sequence for the pre-and pro- regions followed by the coding sequence for the mature mouseneurturin.

Sequence analysis of the murine neurturin genomic clones identified a0.5 kb intron located between nucleotide 169 and 170 of the pre-proneurturin from the cDNA clones. This intron is located in the codingsequence of the pro- region of the pre-pro-neurturin protein. Thus, itis believed that the mouse neurturin gene contains at least two exons,one of which contains the coding sequences upstream from the splice siteand the other contains the coding sequence downstream (FIG. 8, SEQ IDNO:29, SEQ ID NO:30). It is known that the gene for GDNF contains anintron located at an analogous position and an alternately spliced formof GDNF has been detected by RT-PCR experiments (Suter-Crazzolara andUnsicker, Neuroreport 5: 2486-2488, 1994 which is incorporated byreference). This alternate form results from the use of a splice site inthe second coding exon located 78 bp 3' to the original splice sitereported. The alternately spliced form encodes a GDNF protein with adeletion of 26 amino acids relative to the originally reported form. Thetwo forms are expressed in different ratios in different tissues. Wehave not detected alternately spliced forms of neurturin in RT-PCR andRACE experiments using mouse P1 brain and P1 liver cDNAs. Thepossibility exists, however, that alternate splice sites in theneurturin gene may be utilized in different tissues.

The coding sequence of the human neurturin cDNA has been deduced fromthe sequence of the human neurturin genomic clones. The coding sequenceof the human cDNA, like that of the mouse cDNA, is interrupted by anintron between nucleotides 169 and 170 of the coding sequence. Thus, thehuman neurturin gene is believed to contain at least two exons, one ofwhich contains the coding sequence upstream from the splice site and theother contains the coding sequence downstream (FIG. 7, SEQ ID NO:27, SEQID NO:28). The splice sites at the intron-exon junctions of the humanand mouse genes have been conserved.

From the deduced amino acid sequence of human neurturin, the earlierpredicted N-terminal sequence lies between positions 286 and 339 and thepredicted internal sequences lie between positions 385 and 417,positions 474 and 533, and positions 547 and 576. The TGA stop codon atpositions 592-594 terminate the open reading frame.

The predicted length of the purified pre-pro neurturin is 197 amino acidresidues for the human pre-pro neurturin (SEQ ID NO:7) and 195 aminoacid residues for the mouse pre-pro neurturin (SEQ ID NO:8). Thepredicted molecular weight of this polypeptide is 22.2 kD for mouse and22.4 kd for human. The predicted length of the purified neurturin is 100amino acid residues and its predicted monomeric molecular weight is 11.5kD. There are no N-linked glycosolation sites, however, potentialO-linked glycosolation sites occur at amino acid residues in positions18, 26, 80, 86 and 95 in human neurturin. Glycosylation at any one orcombination of these sites would increase the molecular weight of themolecule.

Different possible cleavage sites may be present in thepre-pro-neurturin sequence. The amino acid sequence of the mature mouseneurturin (FIG. 5, SEQ ID NO:2) is predicted from alignment with theN-terminal amino acid sequence of the purified chinese hamsterneurturin. A four residue RRAR cleavage site (amino acids 92-95) isfound immediately before the predicted N-terminal amino acid of maturemouse neurturin. This RRAR sequence fits the RXXR consensus sequence atwhich members of the TGF-β superfamily are usually cleaved. Thisputative RRAR cleavage sequence is conserved in human neurturin.However, the mature human neurturin is predicted to have a two aminoacid N-terminal extension relative to mature mouse neurturin whencleaved at this sequence. Since neurturin contains other sequences whichfit the RXXR consensus (for example the sequence RRRR at amino acids90-93) and the specificities of proteases involved in this cleavage arenot completely understood, the possibility exists that in somesituations, neurturin is cleaved at sites other than the above RRARsequence, and the mature neurturin protein may have a variable number ofamino acids preceding the cysteine residue at position 101 in the mousesequence (pre-pro protein) and position 103 in the human sequence. Suchalternate cleavage sites could be utilized differently among differentorganisms and among different tissues of the same organism. TheN-terminal amino acids preceding the first of the seven conservedcysteines in the mature forms of members of the TGF-β family varygreatly in both length and sequence. Furthermore, insertion of a tenamino acid sequence two residues upstream of the first conservedcysteine does not affect the known biological activities of one familymember, dorsalin (Basler, K., Edlund, T., Jessell,T. M., and Yamada, T.,(1993) Cell 73:687-702). Thus neurturin proteins which contain sequencesof different lengths preceding the cysteine 101 in mouse and cysteine103 in human would be likely to retain their biological activity.

The inventors herein believe that at a minimum the sequence of neurturinthat will show biological activity will contain the sequence beginningat cysteine 103 and ending at cysteine 196 for human neurturin (FIG. 7,SEQ ID NO:31) and beginning at cysteine 101 and ending at cysteine 194for mouse neurturin (FIG. 7, SEQ ID NO:32). Thus, within the scope ofthe present invention are amino acid sequences containing SEQ ID NO:31and amino acid sequences containing SEQ ID NO:32 and nucleic acidsequences encoding these amino acid sequences.

The present invention includes nucleic acid sequences includingsequences that encode human and mouse neurturin (FIG. 5). Also includedwithin the scope of this invention are sequences that are substantiallythe same as the nucleic acid sequences encoding neurturin. Suchsubstantially the same sequences may, for example, be substituted withcodons more readily expressed in a given host cell such as E. coliaccording to well known and standard procedures. Such modified nucleicacid sequences would be included within the scope of this invention.

Specific nucleic acid sequences can be modified by those skilled in theart and, thus, all nucleic acid sequences which encode for the aminoacid sequences of pre-pro neurturin or the pre- region or the pro-region or neurturin can likewise be so modified. The present inventionthus also includes nucleic acid sequence which will hybridize with allsuch nucleic acid sequences--or complements of the nucleic acidsequences where appropriate--and encode for a polypeptide having cellsurvival promoting activity. The present invention also includes nucleicacid sequences which encode for polypeptides that have neuronal survivalpromoting activity and that are recognized by antibodies that bind toneurturin.

The present invention also encompasses vectors comprising expressionregulatory elements operably linked to any of the nucleic acid sequencesincluded within the scope of the invention. This invention also includeshost cells--of any variety--that have been transformed with vectorscomprising expression regulatory elements operably linked to any of thenucleic acid sequences included within the scope of the presentinvention.

Methods are also provided herein for producing neurturin. Preparationcan be by isolation from conditioned medium from a variety of cell typesso long as the cell type produces neurturin. A second and preferredmethod involves utilization of recombinant methods by isolating anucleic acid sequence encoding neurturin, cloning the sequence alongwith appropriate regulatory sequences into suitable vectors and celltypes, and expressing the sequence to produce neurturin.

A mammalian gene family comprised of four neurotrophic factors has beenidentified including nerve growth factor (NGF), brain derivedneurotrophic factor (BDGF), neurotrophin-3 (NT-3), and neurotrophin-4/5(NT-4/5). These factors share approximately 60 percent nucleic acidsequence homology (Tuszynski and Gage, Ann Neurol 35:S9-S12, 1994 whichis incorporated by reference). The neurturin protein displays nosignificant homology to the NGF family of neurotrophic factors.Neurturin shares less than about 20% homology with the TGF-β superfamilyof growth factors. However, neurturin shows approximately 40% sequenceidentity with GDNF. In particular, the positions of the seven cysteineresidues present in both neurturin and GDNF are exactly conserved. Theinventors herein believe that other unidentified genes may exist thatencode proteins that have substantial amino acid sequence homology toneurturin and GDNF and which function as growth factors selective forthe same or different tissues and the same or different biologicalactivities. A different spectrum of activity with respect to tissuesaffected and/or response elicited could result from preferentialactivation of different receptors by different family members as isknown to occur with members of the NGF family of neurotrophic factors(Tuszynski and Gage, 1994, supra).

As a consequence of members of a particular gene family showingsubstantial conservation of amino acid sequence among the proteinproducts of the family members, there is considerable conservation ofsequences at the DNA level. This forms the basis for a new approach foridentifying other members of the gene family to which GDNF and neurturinbelong. The method used for such identification is cross-hybridizationusing nucleic acid probes derived from one family member to form astable hybrid duplex molecule with nucleic acid sequence from differentmembers of the gene family or to amplify nucleic acid sequences fromdifferent family members. (see for example, Kaisho et al. FEBS Letters266:187-191, 1990 which is incorporated by reference). The sequence fromthe different family member may not be identical to the probe, but will,nevertheless be sufficiently related to the probe sequence to hybridizewith the probe. Alternatively, PCR using primers from one family membercan be used to identify additional family members.

The above approaches have not heretofore been successful in identifyingother gene family members because only one family member, GDNF wasknown. With the identification of neurturin herein, however, unique newprobes and primers can be made that contain sequences from the conservedregions of this gene family. In particular, three conserved regions havebeen identified herein which can be used as a basis for constructing newprobes and primers. The new probes and primers made available from thepresent work make possible this powerful new approach which can nowsuccessfully identify other gene family members. Using this newapproach, one may screen for genes related to GDNF and neurturin insequence homology by preparing DNA or RNA probes based upon theconserved regions in the GDNF and neurturin molecules. Therefore, oneembodiment of the present invention comprises probes and primers thatare unique to or derived from a nucleotide sequence encoding suchconserved regions and a method for identifying further members of theGDNF-neurturin gene family. Conserved region amino acid sequencesinclude Val-Xaa₁ -Xaa₂ -Leu-Gly-Leu-Gly-Tyr in which Xaa₁ is Ser or Thrand Xaa₂ is Glu or Asp (SEQ ID NO:33); Glu-Xaa₁ -Xaa₂ -Xaa₃-Phe-Arg-Tyr-Cys-Xaa₄ -Gly-Xaa₅ -Cys-Xaa₆ -Xaa₇ -Ala in which Xaa₁ isThr or Glu, Xaa₂ is Val or Leu, Xaa₃ is Leu or Ile, Xaa₄ is Ala or Ser,Xaa₅ is Ala or Ser, Xaa₆ is Glu or Asp and Xaa₇ is Ala or Ser (SEQ IDNO:34); and Cys-Cys-Arg-Pro-Xaa₁ -Ala-Xaa₂ -Xaa3-Asp-Xaa₄ -Xaa₅-Ser-Phe-Leu-Asp in which Xaa₁ is Thr or Val or Ile, Xaa2 is Tyr or Phe,Xaa₃ is Glu or Asp, Xaa₄ is Glu or Asp and Xaa₅ is Val or Leu (SEQ IDNO:35). Nucleotide sequences containing a coding sequence for the aboveconserved sequences or fragments of the above conserved sequences can beused as probes. Exemplary probe and primer sequences include nucleicacid sequences encoding amino acid sequences, SEQ ID NO:33, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO 39, SEQ ID NO:40 and SEQ IDNO:41 and, in particular, nucleic acid sequences, SEQ ID NOS:42, SEQ IDNOS:43, SEQ ID NOS:44, SEQ ID NOS:45, SEQ ID NOS:46, SEQ ID NOS:47, andSEQ ID NOS:48.

Hybridization using the new probes from conserved regions of the nucleicacid sequences would be performed under reduced stringency conditions.Factors involved in determining stringency conditions are well known inthe art (for example, see Sambrook et al., Molecular Cloning, 2nd Ed.,1989 which is incorporated by reference). Sources of nucleic acid forscreening would include genomic DNA libraries from mammalian species orcDNA libraries constructed using RNA obtained from mammalian cellscloned into any suitable vector.

PCR primers would be utilized under PCR conditions of reduced annealingtemperature which would allow amplification of sequences from genefamily members other than GDNF and neurturin. Sources of nucleic acidfor screening would include genomic DNA libraries from mammalian speciescloned into any suitable vector, cDNA transcribed from RNA obtained frommammalian cells, and genomic DNA from mammalian species.

DNA sequences identified on the basis of hybridization or PCR assayswould be sequenced and compared to GDNF and neurturin. The DNA sequencesencoding the entire sequence of the novel factor would then be obtainedin the same manner as described herein. Genomic DNA or libraries ofgenomic clones can also be used as templates because the intron/exonstructures of GDNF and neurturin are conserved and coding sequences ofthe mature proteins are not interrupted by introns.

Although neurturin has been purified on the basis of its ability topromote the survival of a particular neuronal type, this factor will acton other neuronal cell types as well. For example, neurturin is shownherein to promote the survival of nodose sensory ganglia neurons (seeExample 3). Neurturin is also likely to promote the survival ofnon-neuronal cells. Indeed, all the growth factors isolated to date havebeen shown to act on many different cell types (for example see Scullyand Otten, Cell Biol Int 19:459-469, 1005; Hefti, Neurotrophic FactorTherapy 25:1418-1435, 1994 which are incorporated by reference). It isknown that NGF acts on sympathetic neurons, several types of sensoryneurons and certain populations of CNS neurons. GDNF, which is moreclosely related to neurturin, has been shown to act on dopaminergic,sympathetic, motor and several sensory neurons (Henderson et al. supra,1994; Miles et al, J Cell Biol 130:137-148, 1995; Yan et al, Nature373:341-344, 1995; Lin et al, Science 260:1130-1132, 1993; Trupp et al,J Cell Biol 130:137-148, 1995; Martin et al Brain Res 683:172-178, 1995;Bowenkamp st al J Comp Neurol 355:479-489, 1995 which are incorporatedby reference). Thus, it is likely that in addition to peripheralsympathetic and sensory neurons, neurturin can act on a wide variety ofcentral and peripheral neuronal cell types.

It is also likely that neurturin will act on non-neuronal cells topromote their survival, growth or function. This expectation is basedupon the activity of known growth factors. Although NGF is theprototypical neurotrophic factor, this growth factor also acts upon mastcells to increase the number of mast cells when injected into newbornrats (Aloe, J Neuroimmunol 18:1-12, 1988). In addition, mast cellsexpress the trk receptor and respond to NGF such that NGF is a mast cellsecretogogue and survival promoting factor (Horigome et al., J Bol Chem269:2695-2707, 1994 which is incorporated by reference). Moreover,members of the TGF-β superfamily act on many cell types of differentfunction and embryologic origin.

The inventors herein have identified several non-neuronal tissues inwhich neurturin is expressed including blood, bone marrow, neonatalliver and mast cells. This suggests a role for neurturin inhematopoiesis, inflammation and allergy.

Neurotrophic factors of the NGF family are thought to act throughfactor-specific high affinity receptors (Tuszynski and Gage, 1994,supra). Only particular portions of the protein acting at a receptorsite are required for binding to the receptor. Such particular portionsor discrete fragments can serve as an agonist where the substanceactivates the receptor to elicit the promoting action on cell survivaland growth and antagonists to neurturin where they bind to, but do notactivate, the receptor or promote survival and growth. Such portions orfragments that are agonists and those that are antagonists are alsowithin the scope of the present invention.

Synthetic, pan-growth factors can also be constructed by combining theactive domains of neurturin with the active domains of one or more othergrowth factors. (For example, see Ilag et al., Proc Nat'l Acad Sci92:607-611, 1995 which is incorporated by reference). These pan-growthfactors would be expected to have the combined activities of neurturinand the one or more other growth factors. As such they are believed tobe potent and multispecific growth factors that are useful in thetreatment of a wide spectrum of degenerative diseases and conditionsincluding conditions that can be treated by any and all of the parentfactors from which the active domains were obtained. Such pan-growthfactors might also provide synergistic effects beyond the activities ofthe parent factors (Barres et al., supra).

The present invention also includes therapeutic or pharmaceuticalcompositions comprising neurturin in an effective amount for treatingpatients with cellular degeneration and a method comprisingadministering a therapeutically effective amount of neurturin. Thesecompositions and methods are useful for treating a number ofdegenerative diseases. Where the cellular degeneration involves neuronaldegeneration, the diseases include, but are not limited to peripheralneuropathy, amyotrophic lateral sclerosis, Alzheimer's disease,Parkinson's disease, Huntington's disease, ischemic stroke, acute braininjury, acute spinal chord injury, nervous system tumors, multiplesclerosis, peripheral nerve trauma or injury, exposure to neurotoxins,metabolic diseases such as diabetes or renal dysfunctions and damagecaused by infectious agents. Where the cellular degeneration involvesbone marrow cell degeneration, the diseases include, but are not limitedto disorders of insufficient blood cells such as, for example,leukopenias including eosinopenia and/or basopenia, lymphopenia,monocytopenia, neutropenia, anemias, thrombocytopenia as well as aninsufficiency of stem cells for any of the above. The above cells andtissues can also be treated for depressed function.

The compositions and methods herein can also be useful to preventdegeneration and/or promote survival in other non-neuronal tissues aswell. One skilled in the art can readily determine using a variety ofassays known in the art for identifying whether neurturin would beuseful in promoting survival or functioning in a particular cell type.

In certain circumstances, it may be desirable to modulate or decreasethe amount of neurturin expressed. Thus, in another aspect of thepresent invention, neurturin anti-sense oligonucleotides can be made anda method utilized for diminishing the level of expression of neurturinby a cell comprising administering one or more neurturin anti-senseoligonucleotides. By neurturin anti-sense oligonucleotides reference ismade to oligonucleotides that have a nucleotide sequence that interactsthrough base pairing with a specific complementary nucleic acid sequenceinvolved in the expression of neurturin such that the expression ofneurturin is reduced. Preferably, the specific nucleic acid sequenceinvolved in the expression of neurturin is a genomic DNA molecule ormRNA molecule that encodes neurturin. This genomic DNA molecule cancomprise regulatory regions of the neurturin gene, the pre- or pro-portions of the neurturin gene or the coding sequence for matureneurturin protein. The term complementary to a nucleotide sequence inthe context of neurturin antisense oligonucleotides and methods thereformeans sufficiently complementary to such a sequence as to allowhybridization to that sequence in a cell, i.e., under physiologicalconditions. The neurturin antisense oligonucleotides preferably comprisea sequence containing from about 8 to about 100 nucleotides and morepreferably the neurturin antisense oligonucleotides comprise from about15 to about 30 nucleotides. The neurturin antisense oligonucleotides canalso contain a variety of modifications that confer resistance tonucleolytic degradation such as, for example, modified internucleosidelinaages (Uhlmann and Peyman, Chemical Reviews 90:543-548 1990;Schneider and Banner, Tetrahedron Lett 31:335, 1990 which areincorporated by reference), modified nucleic acid bases and/or sugarsand the like.

The therapeutic or pharmaceutical compositions of the present inventioncan be administered by any suitable route known in the art including forexample intravenous, subcutaneous, intramuscular, transdermal,intrathecal or intracerebral. Administration can be either rapid as byinjection or over a period of time as by slow infusion or administrationof slow release formulation. For treating tissues in the central nervoussystem, administration can be by injection or infusion into thecerebrospinal fluid (CSF). When it is intended that neurturin beadministered to cells in the central nervous system, administration canbe with one or more agents capable of promoting penetration of neurturinacross the blood-brain barrier.

Neurturin can also be linked or conjugated with agents that providedesirable pharmaceutical or pharmacodynamic properties. For example,neurturin can be coupled to any substance known in the art to promotepenetration or transport across the blood-brain barrier such as anantibody to the transferrin receptor, and administered by intravenousinjection (See for example, Friden et al., Science 259:373-377, 1993which is incorporated by reference). Furthermore, neurturin can bestably linked to a polymer such as polyethylene glycol to obtaindesirable properties of solubility, stability, half-life and otherpharmaceutically advantageous properties. (See for example Davis et al.Enzyme Eng 4:169-73, 1978; Burnham, Am J Hosp Pharm 51:210-218, 1994which are incorporated by reference).

The compositions are usually employed in the form of pharmaceuticalpreparations. Such preparations are made in a manner well known in thepharmaceutical art. One preferred preparation utilizes a vehicle ofphysiological saline solution, but it is contemplated that otherpharmaceutically acceptable carriers such as physiologicalconcentrations of other non-toxic salts, five percent aqueous glucosesolution, sterile water or the like may also be used. It may also bedesirable that a suitable buffer be present in the composition. Suchsolutions can, if desired, be lyophilized and stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection. The primary solvent can be aqueous or alternativelynon-aqueous. Neurturin can also be incorporated into a solid orsemi-solid biologically compatible matrix which can be implanted intotissues requiring treatment.

The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dosage ormulti-dose form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

Dose administration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused.

It is also contemplated that certain formulations containing neurturinare to be administered orally. Such formulations are preferablyencapsulated and formulated with suitable carriers in solid dosageforms. Some examples of suitable carriers, excipients, and diluentsinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium,stearate, water, mineral oil, and the like. The formulations canadditionally include lubricating agents, wetting agents, emulsifying andsuspending agents, preserving agents, sweetening agents or flavoringagents. The compositions may be formulated so as to provide rapid,sustained, or delayed release of the active ingredients afteradministration to the patient by employing procedures well known in theart. The formulations can also contain substances that diminishproteolytic degradation and promote absorption such as, for example,surface active agents.

The specific dose is calculated according to the approximate body weightor body surface area of the patient or the volume of body space to beoccupied. The dose will also be calculated dependent upon the particularroute of administration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art in light ofthe activity disclosed herein in assay preparations of target cells.Exact dosages are determined in conjunction with standard dose-responsestudies. It will be understood that the amount of the compositionactually administered will be determined by a practitioner, in the lightof the relevant circumstances including the condition or conditions tobe treated, the choice of composition to be administered, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration.

In one embodiment of this invention, neurturin may be therapeuticallyadministered by implanting into patients vectors or cells capable ofproducing a biologically-active form of neurturin or a precursor ofneurturin, i.e. a molecule that can be readily converted to abiological-active form of neurturin by the body. In one approach cellsthat secrete neurturin may be encapsulated into semipermeable membranesfor implantation into a patient. The cells can be cells that normallyexpress neurturin or a precursor thereof or the cells can be transformedto express neurturin or a precursor thereof. It is preferred that thecell be of human origin and that the neurturin be human neurturin whenthe patient is human. However, the formulations and methods herein canbe used for veterinary as well as human applications and the term"patient" as used herein is intended to include human and veterinarypatients.

Cells can be grown ex vivo for use in transplantation or engraftmentinto patients (Muench et al., Leuk & Lymph 16:1-11, 1994 which isincorporated by reference). In another embodiment of the presentinvention, neurturin is used to promote the ex vivo expansion of a cellsfor transplantation or engraftment. Current methods have used bioreactorculture systems containing factors such as erythropoietin, colonystimulating factors, stem cell factor, and interleukins to expandhematopoietic progenitor cells for erythrocytes, monocytes, neutrophils,and lymphocytes (Verfaillie, Stem Cells 12:466-476, 1994 which isincorporated by reference). These stem cells can be isolated from themarrow of human donors, from human peripheral blood, or from umbilicalcord blood cells. The expanded blood cells are used to treat patientswho lack these cells as a result of specific disease conditions or as aresult of high dose chemotherapy for treatment of malignancy (George,Stem Cells 12(Suppl 1):249-255, 1994 which is incorporated byreference). In the case of cell transplant after chemotherapy,autologous transplants can be performed by removing bone marrow cellsbefore chemotherapy, expanding the cells ex vivo using methods that alsofunction to purge malignant cells, and transplanting the expanded cellsback into the patient following chemotherapy (for review see Rummel andVan Zant, J Hematotherapy 3:213-218, 1994 which is incorporated byreference). Since neurturin is expressed in the developing animal inblood, bone marrow and liver, tissues where proliferation anddifferentiation of progenitor cells occur, it is believed that neurturincan function to regulate the proliferation of hematopoietic stem cellsand the differentiation of mature hematopoietic cells. Thus, theaddition of neurturin to culture systems used for ex vivo expansion-ofcells could stimulate the rate at which certain populations of cellsmultiply or differentiate, and improve the effectiveness of theseexpansion systems in generating cells needed for transplant.

It is also believed that neurturin can be used for the ex vivo expansionof precursor cells in the nervous system. Transplant or engraftment ofcells is currently being explored as a therapy for diseases in whichcertain populations of neurons are lost due to degeneration such as, forexample, in parkinson's disease (Bjorklund, Curr Opin Neurobiol2:683-689, 1992 which is incorporated by reference). Neuronal precursorcells can be obtained from animal or human donors or from human fetaltissue and then expanded in culture using neurturin or other growthfactors. These cells can then be engrafted into patients where theywould function to replace some of the cells lost due to degeneration.Because neurotrophins have been shown to be capable of stimulating thesurvival and proliferation of neuronal precursors cells such as, forexample, NT-3 stimulation of sympathetic neuroblast cells (Birren etal., Develop 119:597-610, 1993 which is incorporated by reference),neurturin could also function in similar ways during the development ofthe nervous system and could be useful in the ex vivo expansion ofneuronal cells.

In a number of circumstances it would be desirable to determine thelevels of neurturin in a patient. The identification of neurturin alongwith the present report showing that neurturin is expressed by a numberof tissues provides the basis for the conclusion that the presence ofneurturin serves a normal physiologic function related to cell growthand survival. Indeed, other neurotrophic factors are known to play arole in the function of neuronal and non-neuronal tissues. (For reviewsee Scully and Otten, Cell Bol Int 19:459-469, 1995; Otten and Gadient,Int J Devl Neurosciences 13:147-151, 1995 which are incorporated byreference). Endogenously produced neurturin may also play a role incertain disease conditions, particularly where there is cellulardegeneration such as in neurodegenerative conditions or diseases. Otherneurotrophic factors are known to change during disease conditions. Forexample, in multiple sclerosis, levels of NGF protein in thecerebrospinal fluid are increased during acute phases of the disease(Bracci-Laudiero et al., Neuroscience Lett 147:9-12, 1992 which isincorporated by reference) and in systemic lupus erythematosus there isa correlation between inflammatory episodes and NGF levels in sera(Bracci-Laudiero et al. NeuroReport 4:563-565, 1993 which isincorporated by reference).

Given that neurturin is expressed in blood cells, bone marrow and mastcells, it is likely that the level of neurturin may be altered in avariety of conditions and that quantification of neurturin levels wouldprovide clinically useful information. Furthermore, in the treatment ofdegenerative conditions, compositions containing neurturin can beadministered and it would likely be desirable to achieve certain targetlevels of neurturin in sera, in cerebrospinal fluid or in any desiredtissue compartment. It would, therefore, be advantageous to be able tomonitor the levels of neurturin in a patient. Accordingly, the presentinvention also provides methods for detecting the presence of neurturinin a sample from a patient.

The term "detection" as used herein in the context of detecting thepresence of neurturin in a patient is intended to include thedetermining of the amount of neurturin or the ability to express anamount of neurturin in a patient, the distinguishing of neurturin fromother growth factors, the estimation of prognosis in terms of probableoutcome of a degenerative disease and prospect for recovery, themonitoring of the neurturin levels over a period of time as a measure ofstatus of the condition, and the monitoring of neurturin levels fordetermining a preferred therapeutic regimen for the patient.

To detect the presence of neurturin in a patient, a sample is obtainedfrom the patient. The sample can be a tissue biopsy sample or a sampleof blood, plasma, serum, CSF or the like. Neurturin is expressed in awide variety of tissues as shown in example 9. Samples for detectingneurturin can be taken from any of these tissues. When assessingperipheral levels of neurturin, it is preferred that the sample be asample of blood, plasma or serum. When assessing the levels of neurturinin the central nervous system a preferred sample is a sample obtainedfrom cerebrospinal fluid.

In some instances it is desirable to determine whether the neurturingene is intact in the patient or in a tissue or cell line within thepatient. By an intact neurturin gene it is meant that there are noalterations in the gene such as point mutations, deletions, insertions,chromosomal breakage, chromosomal rearrangements and the like whereinsuch alteration might alter production of neurturin or alter itsbiological activity, stability or the like to lead to disease processesor susceptibility to cellular degenerative conditions. Thus, in oneembodiment of the present invention a method is provided for detectingand characterizing any alterations in the neurturin gene. The methodcomprises providing an oligonucleotide that contains the neurturin cDNA,genomic DNA or a fragment thereof or a derivative thereof. By aderivative of an oligonucleotide, it is meant that the derivedoligonucleotide is substantially the same as the sequence from which itis derived in that the derived sequence has sufficient sequencecomplementarily to the sequence from which it is derived to hybridize tothe neurturin gene. The derived nucleotide sequence is not necessarilyphysically derived from the nucleotide sequence, but may be generated inany manner including for example, chemical synthesis or DNA replicationor reverse transcription or transcription.

Typically, patient genomic DNA is isolated from a cell sample from thepatient and digested with one or more restriction endonucleases such as,for example, TaqI and AluI. Using the Southern blot protocol, which iswell known in the art, this assay determines whether a patient or aparticular tissue in a patient has an intact neurturin gene or aneurturin gene abnormality.

Hybridization to the neurturin gene would involve denaturing thechromosomal DNA to obtain a single-stranded DNA; contacting thesingle-stranded DNA with a gene probe associated with the neurturin genesequence; and identifying the hybridized DNA-probe to detect chromosomalDNA containing at least a portion of the human neurturin gene.

The term "probe" as used herein refers to a structure comprised of apolynucleotide which forms a hybrid structure with a target sequence,due to complementarity of probe sequence with a sequence in the targetregion. Oligomers suitable for use as probes may contain a minimum ofabout 8-12 contiguous nucleotides which are complementary to thetargeted sequence and preferably a minimum of about 20.

The neurturin gene probes of the present invention can be DNA or RNAoligonucleotides and can be made by any method known in the art such as,for example, excision, transcription or chemical synthesis. Probes maybe labelled with any detectable label known in the art such as, forexample, radioactive or fluorescent labels or enzymatic marker. Labelingof the probe can be accomplished by any method known in the art such asby PCR, random priming, end labelling, nick translation or the like. Oneskilled in the art will also recognize that other methods not employinga labelled probe can be used to determine the hybridization. Examples ofmethods that can be used for detecting hybridization include Southernblotting, fluorescence in situ hybridization, and single-strandconformation polymorphism with PCR amplification.

Hybridization is typically carried out at 25°-45° C., more preferably at32°-40° C. and more preferably at 37°-38° C. The time required forhybridization is from about 0.25 to about 96 hours, more preferably fromabout one to about 72 hours, and most preferably from about 4 to about24 hours.

Neurturin gene abnormalities can also be detected by using the PCRmethod and primers that flank or lie within the neurturin gene. The PCRmethod is well known in the art. Briefly, this method is performed usingtwo oligonucleotide primers which are capable of hybridizing to thenucleic acid sequences flanking a target sequence that lies within aneurturin gene and amplifying the target sequence. The terms"oligonucleotide primer" as used herein refers to a short strand of DNAor RNA ranging in length from about 8 to about 30 bases. The upstreamand downstream primers are typically from about 20 to about 30 basepairs in length and hybridize to the flanking regions for replication ofthe nucleotide sequence. The polymerization is catalyzed by aDNA-polymerase in the presence of deoxynucleotide triphosphates ornucleotide analogs to produce double-stranded DNA molecules. The doublestrands are then separated by any denaturing method including physical,chemical or enzymatic. Commonly, the method of physical denaturation isused involving heating the nucleic acid, typically to temperatures fromabout 80° C. to 105° C. for times ranging from about 1 to about 10minutes. The process is repeated for the desired number of cycles.

The primers are selected to be substantially complementary to the strandof DNA being amplified. Therefore, the primers need not reflect theexact sequence of the template, but must be sufficiently complementaryto selectively hybridize with the strand being amplified.

After PCR amplification, the DNA sequence comprising neurturin orpre-pro neurturin or a fragment thereof is then directly sequenced andanalyzed by comparison of the sequence with the sequences disclosedherein to identify alterations which might change activity or expressionlevels or the like.

In another embodiment a method for detecting neurturin is provided basedupon an analysis of tissue expressing the neurturin gene. Certaintissues such as those identified below in example 9 have been found toexpress the neurturin gene. The method comprises hybridizing apolynucleotide to mRNA from a sample of tissues that normally expressthe neurturin gene. The sample is obtained from a patient suspected ofhaving an abnormality in the neurturin gene or in the neurturin gene ofparticular cells. The polynucleotide comprises SEQ ID NO:11 or aderivative thereof or a fragment thereof.

To detect the presence of mRNA encoding neurturin protein, a sample isobtained from a patient. The sample can be from blood or from a tissuebiopsy sample. The sample may be treated to extract the nucleic acidscontained therein. The resulting nucleic acid from the sample issubjected to gel electrophoresis or other size separation techniques.

The mRNA of the sample is contacted with a DNA sequence serving as aprobe to form hybrid duplexes. The use of a labeled probes as discussedabove allows detection of the resulting duplex.

When using the cDNA encoding neurturin protein or a derivative of thecDNA as a probe, high stringency conditions can be used in order toprevent false positives, that is the hybridization and apparentdetection of neurturin nucleotide sequences when in fact an intact andfunctioning neurturin gene is not present. When using sequences derivedfrom the neurturin cDNA, less stringent conditions could be used,however, this would be a less preferred approach because of thelikelihood of false positives. The stringency of hybridization isdetermined by a number of factors during hybridization and during thewashing procedure, including temperature, ionic strength, length of timeand concentration of formamide. These factors are outlined in, forexample, Sambrook et al. (Sambrook, et al., 1989, supra).

In order to increase the sensitivity of the detection in a sample ofmRNA encoding the neurturin protein, the technique of reversetranscription/polymerization chain reaction (RT/PCR) can be used toamplify cDNA transcribed from mRNA encoding the neurturin protein. Themethod of RT/PCR is well known in the art (see example 9 and FIG. 6below).

The RT/PCR method can be performed as follows. Total cellular RNA isisolated by, for example, the standard guanidium isothiocyanate methodand the total RNA is reverse transcribed. The reverse transcriptionmethod involves synthesis of DNA on a template of RNA using a reversetranscriptase enzyme and a 3' end primer. Typically, the primer containsan oligo(dT) sequence. The cDNA thus produced is then amplified usingthe PCR method and neurturin specific primers. (Belyavsky et al, NuclAcid Res 17:2919-2932, 1989; Krug and Berger, Methods in Enzymology,Academic Press, N.Y., Vol. 152, pp. 316-325, 1987 which are incorporatedby reference).

The polymerase chain reaction method is performed as described aboveusing two oligonucleotide primers that are substantially complementaryto the two flanking regions of the DNA segment to be amplified.

Following amplification, the PCR product is then electrophoresed anddetected by ethidium bromide staining or by phosphoimaging.

The present invention further provides for methods to detect thepresence of the neurturin protein in a sample obtained from a patient.Any method known in the art for detecting proteins can be used. Suchmethods include, but are not limited to immunodiffusion,immunoelectrophoresis, immunochemical methods, binder-ligand assays,immunohistochemical techniques, agglutination and complement assays.(for example see Basic and Clinical Immunology, Sites and Terr, eds.,Appleton & Lange, Norwalk, Conn. pp 217-262, 1991 which is incorporatedby reference). Preferred are binder-ligand immunoassay methods includingreacting antibodies with an epitope or epitopes of the neurturin proteinand competitively displacing a labeled neurturin protein or derivativethereof.

As used herein, a derivative of the neurturin protein is intended toinclude a polypeptide in which certain amino acids have been deleted orreplaced or changed to modified or unusual amino acids wherein theneurturin derivative is biologically equivalent to neurturin and whereinthe polypeptide derivative cross-reacts with antibodies raised againstthe neurturin protein. By cross-reaction it is meant that an antibodyreacts with an antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein binding immunoassaysare well known in the art. Antibodies employed in such assays may beunlabeled, for example as used in agglutination tests, or labeled foruse in a wide variety of assay methods. Labels that can be used includeradionuclides, enzymes, fluorescers, chemiluminescers, enzyme substratesor co-factors, enzyme inhibitors, particles, dyes and the like for usein radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.

Polyclonal or monoclonal antibodies to the neurturin protein or anepitope thereof can be made for use in immunoassays by any of a numberof methods known in the art. By epitope reference is made to anantigenic determinant of a polypeptide. An epitope could comprise 3amino acids in a spacial conformation which is unique to the epitope.Generally an epitope consists of at least 5 such amino acids. Methods ofdetermining the spatial conformation of amino acids are known in theart, and include, for example, x-ray crystallography and 2 dimensionalnuclear magnetic resonance.

One approach for preparing antibodies to a protein is the selection andpreparation of an amino acid sequence of all or part of the protein,chemically synthesizing the sequence and injecting it into anappropriate animal, usually a rabbit or a mouse (See Example 10).

Oligopeptides can be selected as candidates for the production of anantibody to the neurturin protein based upon the oligopeptides lying inhydrophilic regions, which are thus likely to be exposed in the matureprotein.

Antibodies to neurturin can also be raised against oligopeptides thatinclude one or more of the conserved regions identified herein such thatthe antibody can cross-react with other family members. Such antibodiescan be used to identify and isolate the other family members.

Methods for preparation of the neurturin protein or an epitope thereofinclude, but are not limited to chemical synthesis, recombinant DNAtechniques or isolation from biological samples. Chemical synthesis of apeptide can be performed, for example, by the classical Merrifeld methodof solid phase peptide synthesis (Merrifeld, J Am Chem Soc 85:2149, 1963which is incorporated by reference) or the FMOC strategy on a RapidAutomated Multiple Peptide Synthesis system (DuPont Company, Wilmington,Del.) (Caprino and Han, J Org Chem 37:3404, 1972 which is incorporatedby reference).

Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified neurturin protein usually by ELISA or by bioassay based uponthe ability to block the action of neurturin on neurons or other cells.When using avian species, e.g. chicken, turkey and the like, theantibody can be isolated from the yolk of the egg. Monoclonal antibodiescan be prepared after the method of Milstein and Kohler by fusingsplenocytes from immunized mice with continuously replicating tumorcells such as myeloma or lymphoma cells. (Milstein and Kohler Nature256:495-497, 1975; Gulfre and Milstein, Methods in Enzymology:Immunochemical Techniques 73:1-46, Langone and Banatis eds., AcademicPress, 1981 which are incorporated by reference). The hybridoma cells soformed are then cloned by limiting dilution methods and supernatesassayed for antibody production by ELISA, RIA or bioassay.

The unique ability of antibodies to recognize and specifically bind totarget proteins provides an approach for treating an over expression ofthe protein. Thus, another aspect of the present invention provides fora method for preventing or treating diseases involving over expressionof the neurturin protein by treatment of a patient with specificantibodies to the neurturin protein.

Specific antibodies, either polyclonal or monoclonal, to the neurturinprotein can be produced by any suitable method known in the art asdiscussed above. For example, murine or human monoclonal antibodies canbe produced by hybridoma technology or, alternatively, the neurturinprotein, or an immunologically active fragment thereof, or ananti-idiotypic antibody, or fragment thereof can be administered to ananimal to elicit the production of antibodies capable of recognizing andbinding to the neurturin protein. Such antibodies can be from any classof antibodies including, but not limited to IgG, IgA, IgM, IgD, and IgEor in the case of avian species, IgY and from any subclass ofantibodies.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1

This example illustrates the isolation and purification of neurturinfrom CHO cell conditioned medium.

Preparation of CHO cell conditioned medium:

A derivative of DG44 Chinese hamster ovary cells, DG44CHO-pHSP-NGFI-B(CHO) cells, was used (Day et al, J Biol Chem 265:15253-15260, 1990which is incorporated by reference). As noted above, the inventors havealso obtained neurturin in partially purified form from otherderivatives of DG44 Chinese hamster ovary cells. The CHO cells weremaintained in 20 ml medium containing minimum essential medium (MEM)alpha (Gibco-BRL No. 12561, Gaithersburg, Md.) containing 10% fetal calfserum (Hyclone Laboratories, Logan, Utah), 2 mM l-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin and 25nM methotrexate using 150 cm²flasks (Corning Inc., Corning N.Y.). For passage and expansion, mediumfrom a confluent flask was aspirated; the cells were washed with 10 mlphosphate buffered saline (PBS) containing in g/l, 0.144 KH₂ PO₄, 0.795Na₂ HPO₄ and 9.00 NaCl; and the flask was then incubated for 2-3 minuteswith 2 ml 0.25% trypsin in PBS. Cells were then knocked of f the flasksurface, 8 ml of medium were added and cells were triturated severaltimes with a pipette. The cells were split 1:5 or 1:10, incubated at 37°C. under an atmosphere of 5% CO₂ in air and grown to confluence for 3-4days.

The cell culture was then expanded into 850 cm² roller bottles (BectonDickinson, Bedford, Mass.). A confluent 150 cm² flask was trypsinizedand seeded into one roller bottle containing 240 ml of the abovemodified MEM medium without methotrexate. The pH was maintained eitherby blanketing the medium with 5% CO₂ in air or by preparing the mediumwith 25 mM HEPES pH 7.4 (Sigma, St. Louis, Mo.). The roller bottles wererotated at 0.8-1.0 revolutions per minute. Cells reached confluence in 4days.

For collecting conditioned medium, serum-free CHO cell (SF-CHO) mediumwas used. SF-CHO was prepared using 1:1 DME/F12 base medium, which wasprepared by mixing 1:1 (v/v) DMEM (Gibco-BRL product No. 11965,Gibco-BRL, Gaithersburg, Md.) with Ham's F12 (Gibco-BRL product No.11765). The final SF-CHO medium contained 15 mM HEPES pH 7.4 (Sigma, St.Louis, Mo.), 0.5 mg/ml bovine serum albumin (BSA, Sigma, St. Louis Mo.),25 μg/ml heparin, (Sigma, St. Louis, Mo.), 1Xinsulin-transferrin-selenite supplement (bovine insulin, 5 μg/ml; humantransferrin, 5 μg/ml; sodium selenite, 5 ng/ml; Sigma, St. Louis, Mo.),2 mM 1-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Themedium from the confluent roller bottles was removed and the cellswashed once with 30 ml SF-CHO medium to remove serum proteins. Cellswere then incubated at 37° C. for 16-24 hrs in 80 ml SF-CHO medium tofurther remove serum proteins. The 80 ml medium was removed anddiscarded. A volume of 120 ml of SF-CHO medium was added to the flaskand the cells incubated at 37° C. Every 48 hrs thereafter, 120 ml wascollected and replaced with the same volume of SF-CHO medium.

Collected media was pooled and centrifuged at 4° C. in polypropyleneconical tubes to remove cellular debris and the supernatant stored at-70° C. Media was collected 5 times over 10 days to yield a total ofapproximately 600 ml conditioned medium per roller bottle.

Fractions collected from the columns at each stage of purification wereassayed for biological activity using the neuronal survival assay andfor protein content by the dye binding assay of Bradford (Anal Biochem72:248 et seq., 1976 which is incorporated by reference). The total mgof protein in the starting volume, typically 50 liters, of conditionedmedium was determined.

Superior Cervical Ganglion Survival Assay:

The neurotrophic activity of CHO conditioned medium starting materialand at various stages of purification was assessed using the superiorcervical ganglion survival assay system previously reported (Martin, etal J of Cell Biology 106:829-844; Deckwerth and Johnson, J Cell Bio123:1207-1222, 1993 which are incorporated by reference). Primarycultures of sympathetic neurons from superior cervical ganglion (SCG)were prepared by dissecting tissue from Day 20-21 rat embryo (E20-E21).The SCG's were placed in Leibovitz's L15 with 1-glutamine medium (Cat#11415-023 Gibco-BRL, Gaithersburg, Md.), digested for 30 minutes with 1mg/ml collagenase (Cat #4188 Worthington Biochemical, Freehold, N.J.) inLeibovitz's L15 medium at 37° C., followed by a 30 minute digestion intrypsin-lyophilized & irradiated (Type TRLVMF Cat #4454 WorthingtonBiochemical, Freehold, N.J.) which was resuspended in modified Hanks'Balanced Salt Solution (Cat #H-8389 Sigma Chemical Co., St. Louis, Mo.).The digestion was stopped using AM50 which contains Minimum EssentialMedium with Earle's salts and without l-glutamine (Cat #11090-016Gibco-BRL), 10% fetal calf serum (Cat #1115 Hyclone Laboratories, Logan,Utah), 2mM l-glutamine (Cat #G5763 Sigm Chemical Co., St. Louis, Mo.),20 μM FuDr (F-0503 Sigma Chemical Co., St. Louis, Mo.), 20 μM Uridine(Cat #3003 Sigma Chemical Co., St. Louis, Mo.), 100 U/ml penicillin, 100μg/ml Streptomycin, and 50 ng/ml 2.5 S NGF. The cells were dissociatedinto a suspension of single cells using a silanized and flame-polishedPasteur pipet. After filtration of the suspension through a nitex filter(size 3-20/14, Tetko Inc., Elmsford, N.Y.), the cells were placed inAM50 medium as above and preplated on a 100 mm Falcon or Primariaculture dish (Becton Dickinson Labware, Lincoln Park, N.J.) to reducethe number of non-neuronal cells. After 2 hours, the medium containingthe unattached neuronal cells was removed from these dishes andtriturated again through a silanized and flame-polished Pasteur pipet.The single cell suspension was plated on 24-well tissue culture plates(Costar, Wilmington, Mass.) that have been previously coated with adouble layer of collagen, one layer of collagen that had been ammoniatedand a second layer of collagen that had been air dried. They wereallowed to attach for 30 minutes to 2 hours. A specific number of viablecells, usually about 1200 to about 3000 total cells per well, or aspecific percentage of the ganglion, usually 25% of the cells obtainedper ganglion were plated into each well. When cell counts were to beperformed they were placed in the 24-well dishes as stated above oralternatively, on 2-well chamber slides (Nunc, Naperville, Ill.).Cultures were then incubated for 5-6 days at 37° in AM50 medium in a 5%CO₂ /95% air atmosphere. The death of the cultured neurons was inducedby exchanging the medium with medium without NGF and with 0.05% goatanti-NGF (final titer in the wells is 1:10). This NGF-deprivationresults in death of the neurons over a period of 24-72 hours. Aliquotsof partially purified or purified factor, or appropriate controls, wereadded to the cultures at the time of NGF removal to determine theability to prevent the neuronal death.

Evaluation of the ability of column fraction, gel eluates, or purifiedfactor to prevent neuronal death was by visual inspection of culturesunder phase contrast microscopy. Viable neurons remained phase brightwith intact neurities, whereas dead neurons were shrunken, phase dark,had irregular membranes and neurites were fragmented (FIG. 3). Whereprecise quantitation of neuronal survival was required, the cultureswere fixed in 4% paraformaldehyde or 10% Formalin in PBS, and stainedwith crystal violet solution, (Huntoon Formula Harleco E. M. DiagnosticsSystems, Gibbstown, N.J.). When using 24 well dishes, 1 μl crystalviolet solution was added to each well containing 10% formalin and thecells were counted using a phase contrast microscope. If the 2-wellchamber slides were used, the cultures were fixed, stained with crystalviolet, destained with water, dehydrated in increasing ethanolconcentrations to toluene, and mounted in a toluene-based mountingsolution. Neurons were scored as viable if they had a clear nucleolusand nuclei and were clearly stained with crystal violet.

The neuronal death at 72 hours in shown in FIG. 3B. Also shown are (A)the positive control cells maintained with nerve growth factor and (C)the cells treated with anti-NGF and neurturin (approximately 3 ng/ml)showing survival of neurons.

Activity was quantitated by calculation of a "survival unit". The totalsurvival units in a sample were defined as the minimal volume of analiquot of the sample which produced maximal survival divided into thetotal volume of that sample. Specific activity was calculated as thesurvival units divided by the mg total protein.

Survival units were determined in an assay using approximately 1200viable neurons in a 0.5 ml culture assay and a culture period of 48hours following addition of the fraction. Survival was assessed visuallyafter the 48 hours. Intrinsic activity as shown in FIG. 4 was determinedin an assay using approximately 2700 neurons and a culture period of 72hours. Survival was assessed by fixing the neurons and counting thenumber of surviving neurons. Because the stability, as assessed byhalf-life of activity, for neurturin decreases as the number of neuronsincreases, the intrinsic activity measurement would be expected to belower than that predicted by Specific Activity determinations. Theintrinsic activity measurement would also be expected to be lower thanthat predicted by specific activity because the survival was measuredafter 72 hours instead of 48 hours.

To ensure the reproducibility of these activity unit assays, it wasnecessary to plate the primary neuronal cultures at reproducible celldensities, as the stability of the activity decreases significantly withincreasing neuronal density. The range of cell densities was from about1200 to about 2700 cells per well. The presence of soluble heparin inthe assay medium had no effect on the short-term (˜3 days) stability ofthe survival activity.

Purification of Neurturin:

Pooled conditioned medium was filtered through 0.2 μl pore bottle-topfilters (cellulose acetate membrane, Corning Inc., Corning, N.Y.).Typically 50-liters of conditioned medium was used and processed in 25liter batches. Each 25 liter batch was introduced at a rate of 20 ml/minonto a 5×5 cm column containing 100 ml heparin-agarose (Sigma, St.Louis, Mo.) equilibrated with 25 mM HEPES, pH 7.4 buffer with 150 mMNaCl. The column was then washed with approximately 1000 ml 25 mM HEPES,pH 7.4 buffer containing 0.5 NaCl at 20 ml/min and the activity was theneluted with 25 mM HEPES, pH 7.4 buffer containing 1.0M NaCl. Afterswitching to the 1.0M NaCL elution buffer, the first 50 ml of buffer wasdiscarded and, thereafter, one 300 ml fraction was collected.

Pooled material eluted from the Heparin-agarose column was then diluted1:1 (v/v) with 25 mM HEPES, pH 7.4 buffer containing 0.04% TWEEN 20 to aNaCl concentration of 0.5M and introduced into a 1.5 cm×9 cm columncontaining 16 ml SP SEPHAROSE® High Performance ion exchange resin(Pharmacia, Piscataway, N.J.) equilibrated in 25 mM HEPES 7.4 containing0.5M NaCl and 0.02% TWEEN 20. The column was then washed with 160 ml 25mM HEPES, pH 7.4 buffer containing 0.5M NaCl and 0.02% TWEEN 20 and theactivity was eluted with 25 mM HEPES, pH 7.4 buffer containing 1.0M NaCland 0.02% TWEEN 20 at a flow rate of 2 ml/min. One 50 ml fraction wascollected after the first 7 ml of eluate from the column.

Material eluted from the SP SEPHAROSE® column was fractionated usingfast protein liquid chromatography (FPLC) on a Chelating Superose HR10/2 column charged with Cu⁺⁺ (Pharmacia, Piscataway, N.J.). The columnhad been prepared by washing with 10 ml water, charging with 3 ml of 2.5mg/ml CuSO₄.5H₂ O, washing with 10 ml water, and equilibrating with 10ml of 25 mM HEPES pH 7.4 buffer containing 1.0M NaCl and 0.02% TWEEN 20.The eluate was introduced into the column in 25 mM HEPES, pH 7.4 buffercontaining 1.0M NaCl at a rate of 1.0 ml/min. The bound proteins wereeluted with a linear gradient of increasing glycine concentration (0-300mM) in 25 mM HEPES, pH 7.4 buffer containing 1.0M NaCl at a rate of 1.0ml/min. The gradient was produced by a Pharmacia FPLC system using anLCC-500 controller and P-500 pumps to establish a 0-300 mM glycinegradient in 40 ml at 1.0 ml/min, thus increasing the gradient by 7.5 mMglucine per min. One ml fractions were collected ,nd assayed for SCGsurvival promotion. Peak activity was observed in fractions 17-20, i.e.17-20 min or ml from the start of the gradient.

Absorbance measurements at 280 nM by an in-line UV monitor indicatedthat most proteins eluted prior to the survival activity in fractions17-20. Thus, significant purification was achieved at this step. A 25 kDband co-purified with the survival activity.

The combined eluted fractions from the Cu⁺⁺ superose column were dilutedto 0.45M NaCl using 25 mM HEPES pH 7.4 buffer containing 0.02% TWEEN 20and introduced into a Mono S HR 5/5 cation exchange column (Pharmacia,Piscataway, N.J.) for further FPLC purification. The column had beenequilibrated with 25 mM HEPES pH 7.4 buffer containing 0.45M NaClcontaining 0.02% TWEEN 20. Bound proteins were eluted with a lineargradient of increasing NaCl concentration (0.45-1.0M). The gradient wasproduced as described-above from 0.45M-1.0M NaCl in 35 mls at 1.0ml/min, thus increasing concentration at 0.0157M per ml or min. Thirteen1.0 ml fractions (fractions 1-13) were collected followed by 44 0.5 mlfractions (fractions 14-53). Peak activity in SCG assay was in fractions26-29. Each fraction was assayed in the SCG survival assay over a rangeof volumes of from 0.1 to 1.0 μl per 0.5 ml culture medium.

One percent (5 μl) of each fraction was loaded onto a non-reducing, 14%SDS polyacrylamide gel and electrophoresed for 750 V-hr at 25° C.Proteins were visualized by silver stain. The results are shown in FIG.2. Markers shown in lane M on the-gel represent 20 ng of Bovine serumalbumin, carbonic anhydrase, B-lactoglobulin, and lysozyme in the orderof descending molecular weight.

A 25 kD band appeared in fractions 25-30, a 28 kD protein elutes earlierin the gradient and an 18 kD elutes later in the gradient. FIG. 2illustrates the survival activity in each of the fractions. The survivalactivity is noted to correspond with the presence and apparent intensityof the 25 kD protein in fractions 25-30.

To demonstrate that the 25 kD band was responsible for survivalpromoting activity, the 25 kD protein was eluted from the polyacrylamidegel after electrophoresis and assayed for survival activity in the SCGassay. After electrophoresis of 150 μl of the SP SEPHAROSE® 1.0M NaClfraction in one lane of a non-reducing 14% SDS-polyacrylamide gel asabove, the lane was cut into 12 slices and each slice was crushed andeluted by diffusion with rocking in buffer containing 25 mM HEPES, pH7.4, 0.5M NaCl, 0.02% Tween-20 for 18 hr at 25° C. BSA was added to theeluate to a final concentration of 200 μg/ml and the eluate was filteredthrough a 0.45 micron filter to remove acrylamide gel fragments. Thefiltrate was then added to a SP SEPHAROSE® column to concentrate andpurify the sample. Before eluting the sample, the column was washed oncein 400 μl 25 mM HEPES, pH 7.4 buffer containing 0.5M NaCl, 0.02%Tween-20 and 200 μg BSA per ml and once in 400 μl 25 mM HEPES, pH 7.4buffer containing 0.02% Tween-20 and 200 μg BSA per ml. The column wasthen washed again in 400 μl of 25 mM HEPES, pH 7.4 buffer containing0.5M NaCl, 0.02% TWEEN 20 and 200 ug BSA per ml. The sample was elutedwith 25 mM HEPES, pH 7.4 buffer containing 1.0M NaCl, 0.02% Tween-20 and200 μg BSA per ml. Samples were then analyzed for survival activity.Only the slice corresponding to the 25 kD band showed evidence ofsurvival activity. The 25 kD protein purified from CHO cell conditionedmedia is believed to be a homodimer.

The yield from the purification above was typically 1-1.5 μg from 50liters of CHO cell conditioned medium. Overall recovery is estimated tobe 10-30%, resulting in a purification of approximately 390,000 fold.

EXAMPLE 2

This example illustrates the characterization of neurturin and severalmembers of the TGF-β family of growth factors in the SCG assay and thelack of cross reactivity of anti-GDNF antibodies with neurturin.

The SCG assay of the purified protein indicated that the factor ismaximally active at a concentration of approximately 3 ng/ml orapproximately 100 pM and the EC₅₀ was approximately 1.5 ng/ml orapproximately 50 pM in the expected range for a diffusible peptidegrowth factor (FIG. 4).

Several members of the TGF-β family influence neuropeptide geneexpression in sympathetic neurons, while others promote survival ofdifferent neuronal populations. Neurturin, which is a distant member ofthis family of proteins, is capable of promoting virtually completesurvival of sympathetic neurons for 3 days. In addition, furtherculturing of the SCG cells revealed that neurturin could continue tomaintain these neurons for at least 10 days after withdrawal of NGF.

We tested several other members of the TGF-β family for their ability topromote survival in the SCG assay including TGF-β1, activin, BMP-2,BMP-4, BMP-6 and GDNF. Of these factors, only GDNF had survivalpromoting activity, however, the activity of GDNF was much less potentthan neurturin in this activity showing an EC₅₀ of 2-4 nM in the 3-daysurvival assay. The GDNF tested in this assay was rhGDNF produced in E.Coli obtained from Prepro Tech, Inc., Rocky Hill, N.J. The duration ofaction of GDNF was also less than that of neurturin inasmuch as theability of GDNF (50 ng/ml) to maintain survival longer than 3 days wassubstantially diminished. These experiments suggest the possibility thatGDNF is a weak agonist for the neurturin receptor. Furthermore, theinability of activin and BMP-2 to promote survival, in contrast to theirstrong induction of transmitter-related gene expression in these neurons(Fann and Paterson, Int J Dev Neurosci 13:317-330, 1995; Fann andPatterson, J Neurochem 61:1349-1355, 1993) suggests that they signalthrough alternate receptors or signal transduction pathways.

To determine the cross-reactivity of anti-GDNF antibodies with partiallypurified neurturin, SCG neurons, that had been dissected and plated asdescribed in Example 1 were treated on Day 6 with 1 ng/ml, 3 ng/ml, 10ng/ml, or 30 ng/ml GDNF (Prepro Tech, Inc, Rocky Hill, N.J.) in thepresence of anti-NGF alone, or in the presence of anti-NGF and anti-GDNF(goat IgG antibody to E. coli-derived rhGDNF, R & D Systems,Minneapolis, Minn.). A partially purified 1.0M SP Sepharose fraction ofneurturin was used in the assay at the approximate concentrations of 375pg/ml, 750 pg/ml, 1.5 ng/ml and 3 ng/ml. This fraction was tested in thepresence of anti-NGF alone, and in the presence of anti-NGF andanti-GDNF. The anti-GDNF antibody blocked the survival promotingactivity of GDNF at a concentration up to 30 ng/ml, but did not blockthe survival promoting activity of neurturin.

EXAMPLE 3

This example illustrates the effect of neurturin on sensory neurons in anodose ganglion survival assay.

CHO cell conditioned media that had been partially purified on the SPSepharose column was assayed for neurotrophic activity on sensoryneurons using nodose ganglia. The survival assay is a modification ofthat previously reported above for superior cervical ganglia. Primarydissociated cultures of nodose ganglia were prepared by dissectingtissue from E18 Sprague Dawley rat pups. The nodose ganglia were placedin Leibovitz's L15 with 2 mM l-glutamine (Cat# 11415-023, GIBCO-BRL.Gaithersburg, Md.) as the tissues was dissected, digested for 30 minwith 1 mg/ml collagenase (Cat#4188, Worthington Biochemical, Freehold,N.J.) in Leibovitz's L15 medium at 37° C., followed by 30 min digestionin trypsin (lyophilized and irradiated, type TRLVMF, Cat #4454Worthington Biochemical, Freehold, N.J.), and resuspension to a finalconcentration of 0.25% in modified Hank's Balanced Salt Solution(Cat#H8389, Sigma Chemical Co., St. Louis, Mo.). The digestion wasstopped using AMO-BDNF100, a medium containing Minimum Essential Mediumwith Earle's salts and without l-glutamine (#11090-016 GIBCO-BRL), 10%fetal Calf Serum (Cat#1115, Hyclone Laboratories, Logan, Utah), 2 mMl-glutamine (Cat#G5763 Sigma Chemical Co., St. Louis, Mo.), 20 μM FuDr(F-0503, Sigma Chemical Co.), 20 μM Uridine (Cat #3003, Sigma ChemicalCo., St. Louis, Mo.) 100 U/ml penicillin, 100 μg/ml Streptomycin, and100 ng Brain Derived Neurotropic Factor (BDNF, Amgen, Thousand Oaks,Calif.). The cells were dissociated into a suspension of single cellsusing a silanized and flame-polished Pasteur pipet in the AMO-BDNF100medium, and preplated on a 100 mm Falcon or Primaria culture dish(Becton Dickinson Labware, Lincoln Park, N.J.) to remove non-neuronalcells. After 2 hours, the medium containing the unattached neuronalcells was removed from these dishes and triturated again through asilanized and flame-polished Pasteur pipet. The single cell suspensionwas plated on 24-well tissue culture plates (Costar, Wilmington, Mass.)that have been previously coated with a double layer of collagen, onelayer of which had been ammoniated and a second layer that had been airdried. Ganglia from ten E18 rat embryos were dissociated into 2.5 mls ofmedia and 100 μl of this suspension was added to each well. The cellswere allowed to attach for 30 min in a 37° C. incubator with 5% CO2/95%air. The wells were fed with AM0-BDNF100 media overnight.

The next day the cells were washed 3 times for 20 min each time with AM0medium containing no BDNF. The wells were fed with 0.5 ml of this mediaalone or this media containing either 50 ng/ml NGF, 100 ng/ml BDNF(Amgen, Thousand Oaks, Calif.), 100 ng/ml GDNF (Prepro Tech, Inc., RockyHill, N.J.) or 3 ng/ml Neurturin. The cells were incubated at 37° C. ina 5% CO₂ /95% air incubator for 3 days, fixed with 10% formalin, stainedwith crystal violet (1 μl/ml 10% formalin) and counted. Survival wasascertained as noted previously.

The neuronal Death at 72 hours is shown in FIG. 10. Neuronal survival ofnodose neurons cultured in BDNF has been previously reported (Thaler etal, Develop Blol 161:338-344, 1994 which is incorporated by reference).This was used as the standard for survival for these neurons and giventhe value of 100% survival. Nodose ganglia that had no trophic support(AM0) showed 20%-30% survival, as did neurons that were cultured in thepresence of 50 ng/ml NGF. Neurons cultured in the presence of 3 ng/mlneurturin and absence of BDNF showed survival similar to those neuronscultured in the presence of BDNF (100 ng/ml). GDNF at a concentration of100 ng/ml promoted greater survival of nodose neurons than did BDNF (100ng/ml). Similar findings with GDNF were recently reported for sensoryneurons from chicken (Ebendal, T. et al, J Neurosci Res 40:276-284 1995which is incorporated by reference).

EXAMPLE 4

This example illustrates the determination of partial amino acidsequences of neurturin isolated from CHO cell conditioned medium.

To obtain N-terminal amino acid sequence from a purified preparation ofapproximately 1 μg of neurturin, the Mono S fractions 26-29 containingthe peak of activity were concentrated to 25 μl by centrifugeultrafiltration in a microcon-3 concentrators (Amicon, Inc., Beverley,Mass.) and loaded onto a non-reducing 14% SDS polyacrylamide gel. Afterelectrophoretic separation, proteins were electroblotted to a PVDFmembrane (Bio-Rad, Hercules, Calif.) and stained with 0.1% CoomassieBlue. The 25 kD band was excised and inserted into the reactioncartridge of an automated sequencer (Model 476, Applied Biosystems(Foster City, Calif.). Phenylthiohydantoin-amino acid (PTH-aa) recoveryin the first 2-3 cycles of automated sequencing by Edman degradationindicated a sequencing yield of 4 pmoles, which was approximately 10% ofthe estimated amount of protein loaded on the SDS gel.

Two N-terminal sequencing runs were performed from two 50 literpurification preparations. In the first run, 1 μg of protein in 3 pooledfractions of 1.5 ml total volume were concentrated to 25 μl andelectroblotted at 100V for 2 hrs at 25° C. using an electroblot bufferof 10 mM CAPS pH 11.0 buffer (Sigma, St. Louis, Mo.) containing 5%methanol. The amino acid sequence was obtained from 13 cycles of Edmandegradation and the sequencing yield was 4 pmoles as above.

In the second run, 1.5 μg of protein in 4 pooled fractions of 2.0 mltotal volume were concentrated to 25 μl and electroblotted at 36V for 12hours at 4° C. using an electroblot buffer of 25 mM Tris, 192 mMglycine, 0.04% SDS and 17% MeOH. Sequencing yield was 15 pmoles and thesequence after 16 cycles was SGARPXGLRELEVSVS (SEQ ID NO:3). Thesequence obtained after 16 cycles corresponded to the shorter sequenceobtained in the first run. Definite assignments could not be made at 3of the amino acid residues in the sequence (residues 1, 6 and 11 fromthe N-terminal). A search of protein databases did not detect anysignificantly homologous sequences, suggesting that the purified factorwas a novel protein.

This initial N-terminal amino acid sequence data did not enable theisolation of cDNA clones using degenerate oligonucleotides as PCRprimers or probes for screening libraries. To facilitate theseapproaches, additional protein was purified in order to obtain internalamino acid sequence from proteolytic fragments. To obtain internal aminoacid sequence from neurturin, an additional 50 liters of CHO cellconditioned medium was purified using only the first 3 chromatographicsteps as outlined above, except that the gradient used to elute the Cu⁺⁺Chelating Superose column was as follows: 0-60 mM glycine (4 ml), 60 mMglycine (10 ml), 60-300 mM glycine (32 ml). Fractions No. 20-23containing neurturin were concentrated to 25 μl by ultrafiltration(Amicon microcon 3, Amicon, Beverley, Mass.) and loaded on anon-reducing SDS polyacrylamide gel. After electrophoresis, the gel wasstained with Coomassie blue and the 25 kD neurturin band was excised.Neurturin was digested in the gel slice with endoproteinase Lys-C, andthe eluted proteolytic fragments were purified by reverse phase HPLC.Only one peak was observed upon HPLC separation of the eluted peptides,which yielded amino acid sequence information for 23 cycles at the 1pmole signal level using the automated sequencer, (internal fragment P2,SEQ ID NO:5).

Amino acid analysis performed on 10% of the above sample beforesubjecting it to digestion had indicated that 150 pmoles of protein werepresent in the gel slice, consisting of 7.6% lysine and 19.5% arginine.The single low level peak from the Lys-C digestion suggested that thedigestion and elution of peptides were inefficient. The same gel slicewas redigested with trypsin and the eluted peptides separated by HPLC.Two peaks were observed on HPLC, resulting in the elucidation of twoadditional 10 residue amino acid sequences (4-5 pmole signal level,internal fragment P1, SEQ ID NO:4 and internal fragment P3, SEQ ID NO:6)that were distinct from the N-terminal and previous internal amino acidsequences. The in situ digestion, elution and purification of peptides,and peptide sequencing was performed by the W. M. Keck FoundationBiotechnology Resource Laboratory at Yale University according tostandard protocols for this service.

EXAMPLE 5

The following example illustrates the isolation and sequence analysis ofmouse and human neurturin cDNA clones.

Degenerate oligonucleotides corresponding to various stretches ofconfident amino acid sequence data were synthesized and used as primersin the polymerase chain reaction (PCR) to amplify cDNA sequences fromreverse transcribed mRNA. A forward primer (M1676; 5'-CCNACNGCNTAYGARGA,SEQ ID NO:50) corresponding to peptide sequence P2 Xaa₁ -Xaa₂-Val-Glu-Ala-Lys-Pro-Cys-Cys-Gly-Pro-Thr-Ala-Tyr-Glu-Asp-Xaa₃-Val-Ser-Phe-Leu-Ser-Val where Xaa₁ and Xaa₂ were unknown, Xaa₃ was Glnor Glu (SEQ ID NO:5) in combination with a reverse primer (M1677;5'-ARYTCYTGNARNGTRTGRTA (SEQ ID NO:52) corresponding to peptide sequenceP3 (Tyr-His-Thr-Leu-Gln-Glu-Leu-Ser-Ala-Arg) (SEQ ID NO:6) were used toamplify a 69 nucleotide product from cDNA templates derived from E21 ratand adult mouse brain. The PCR parameters were: 94° C. for 30 sec; 55°C. for 30 sec; 72° C. for 1 min for 35 cycles. The product was subclonedinto the Bluescript KS plasmid and sequenced. All nucleotide sequencingwas performed using fluorescent dye terminator technology permanufacturer's instructions on an Applied Biosystems automated sequencerModel #373 (Applied Biosystems, Foster City, Calif.). Plasmid DNA forsequencing was prepared using the Wizard Miniprep kit (Promega Corp.,Madison, Wis.) according to the manufacturer's instructions. Thesequence of the amplified product correctly predicted amino acidsequence data internal to the PCR primers.

Primers corresponding to the amplified sequence were used in combinationwith the degenerate primers in the rapid amplification of cDNA ends(RACE) technique (Frohman, M. A. Methods in Enzymology 218:340-356,1993) using the Marathon RACE kit (CLONTECH, Palo Alto, Calif.) per themanufacturer's instructions, except that first strand cDNA synthesis wascarried out at 50° C. using Superscript II reverse transcriptase(Gibco-BRL). Briefly, a double stranded adaptor oligonucleotide wasligated to the ends of double stranded cDNA synthesized from postnatalday 1 rat brain mRNA. Using nested forward neurturin PCR primers (M1676;5'-CCNACNGCNTAYGARGA, SEQ ID NO:50 and 1678;5'-GACGAGGGTCCTTCCTGGACGTACACA, SEQ ID NO:53) in combination withprimers to the ligated adaptor supplied in the kit (AP1, AP2), the 3'end of the neurturin cDNA was amplified by two successive PCR reactions(1st: M1676 and AP1, using 94° C. for 30 sec, 55° C. for 30 sec and 72°C. for 2 min for 35 cycles; 2nd: M1678 and AP2 using 94° C. for 30 secand 68° C. for 2 min for 35 cycles). A 5' portion of the rat neurturincDNA was obtained by two successive PCR reactions using the TinkeredcDNA as template. The 1st reaction utilized primers M1677 (SEQ ID NO:52)and AP1; using 94° C. for 30 sec; 55° C. for 30 sec; and 72° C. for 2min for 35 cycles. The 2nd reaction used M16795'-TAGCGGCTGTGTACGTCCAGGAAGGACACCTCGT (SEQ ID NO:54) and AP2 at 94° C.for 30 sec and 68° C. for 2 min for cycles. These reactions resulted ina truncated form of the 5' end of the neurturin cDNA, apparently theresult of premature termination of the cDNA during reversetranscription. The 5' and 3' RACE products were subcloned into theplasmid Bluescript KS and sequenced. The sequence of these 3' and 5'RACE products resulted in a partial rat neurturin cDNA sequence of 220nt. Primers (#467921 5'-CAGCGACGACGCGTGCGCAAAGAGCG, SEQ ID NO:55; andM1679 (SEQ ID NO:54) corresponding to the partial rat cDNA sequence wereused (PCR parameters 94° C. for 30 sec and 68° C. for 1 min for 35cycles) to amplify a 101 nucleotide PCR product from mouse genomic DNAwhich was homologous to rat neurturin cDNA sequence.

These primers were then used to obtain murine neurturin genomic clonesfrom a mouse 129/Sv library in a P1 bacteriophage vector (libraryscreening service of Genome Systems, Inc., St. Louis, Mo.). A 1.6 kb NcoI fragment from this P1 clone containing the neurturin gene wasidentified by hybridization with primer (#465782;5'-TAYGARGACGAGGTGTCCTTCCTGGACGTACACAGCCGCTAYCAYAC, SEQ ID NO:56). ThisNco I fragment was sequenced and found to contain a stretch of codingsequence corresponding to the N-terminal and internal amino acidsequences obtained from sequencing the active protein isolated from CHOcell conditioned media. Beginning at the N-terminal amino acid sequenceof the purified protein, this nucleotide sequence encodes a 100 aminoacid protein with a predicted molecular mass of 11.5 kD. A search ofprotein and nucleic acid databases identified neurturin as a novelprotein that is approximately 40% identical to glial derivedneurotrophic factor (GDNF). GDNF was purified and cloned as a factorwhich promotes the survival of midbrain dopaminergic neurons and is adistantly related member of the TGF-β superfamily, which now includesmore than 25 different genes that possess a wide variety ofproliferative and differentiative activities. Although GDNF is less than20% identical to any other member of the TGF-β family, it contains the 7cysteine residues which are conserved across the entire family andbelieved to be the basis of a conserved cysteine knot structure observedin the crystal structure determination of TGF-β. Neurturin also containsthese 7 cysteine residues, but like GDNF is less than 20% homologous toany other member of the TGF-β family. Thus, neurturin and GDNF appear torepresent a subfamily of growth factors which have significantlydiverged from the rest of the TGF-β superfamily.

To determine the sequence of the full length mouse neurturin cDNA, 5'and 3' RACE PCR was performed as above for the rat, using nested primerspredicted from the mouse genomic sequence and cDNA from neonatal mousebrain. The 1st reaction for the 3' end used primers: M17775'-GCGGCCATCCGCATCTACGACCGGG (SEQ ID NO:57) and AP1 at 94° C. for 30sec; 65° C. for 15 sec; and 68° C. for 2 min for 35 cycles. The 2ndreaction used primer #467921 (SEQ ID NO:55) and AP2 at 94° C. for 30sec; 65° C. for 15 sec; and 68° C. for 2 min for 20 cycles. The 5' endwas obtained using for the 1st reaction primer M1759,5'-CRTAGGCCGTCGGGCGRCARCACGGGT (SEQ ID NO:58) and AP1 at 94° C. for 30sec; 65° C. for 15 sec; and 68° C. for 2 min for 35 cycles. The 2ndreaction used primer M1785, 5'-GCGCCGAAGGCCCAGGTCGTAGATGCG (SEQ ID NO:59) and AP2 at 94° C. for 30 sec; 65° C. for 15 sec; and 68° C. for 2min for 20 cycles. Both sets of PCR reactions included 5% DMSO. The 5'and 3' mouse RACE products were subcloned into the plasmid Bluescript KSand sequenced. Using the sequence of RACE products, a 1.0 kb mouseneurturin cDNA sequence can be assembled. This cDNA sequence contains anopen reading frame of 585 nucleotides that encodes a protein with amolecular mass of 24 kD. This full length mouse cDNA sequence is shownin FIG. 7 (SEQ ID NO:12). Consistent with the processing events known tooccur for TGF-β family members, the 24 kD neurturin protein contains anamino terminal 19 amino acid signal sequence followed by a pro-domainwhich contains an RXXR proteolytic processing site immediately beforethe N-terminal amino acid sequence obtained when sequencing the proteinpurified from CHO cell conditioned media. Using these landmarks, the11.5 kD mature neurturin molecule is predicted to be 11.5 kD and, byanalogy to other members of the TGF-β family, is predicted to form adisulfide linked homodimer of 23 kD, consistent with the 25 kD mass ofthe protein purified from CHO cell conditioned media as estimated bySDS-PAGE analysis.

For isolation of human genomic clones, primers (#467524;5'-CGCTACTGCGCAGGCGCGTGCGARGCGGC, SEQ ID NO:60 and #10005,5'-CGCCGACAGCTCTTGCAGCGTRTGGTA, SEQ ID NO:61) predicted from thesequence of mouse neurturin were used to amplify (PCR parameters:Initial denaturation at 95° C. for 1 min 30 sec followed by 94° C. for30 see; 60° C. for 15 see; and 68° C. for 60 see for 35 cycles) a 192nucleotide fragment from human genomic DNA. The sequence of the PCRproduct demonstrated that it was the human homolog of mouse neurturin.The primers were then used to screen a human genomic library constructedin the P1 vector (library screening service, Genome Systems, Inc.) andtwo clones containing the human neurturin genomic locus were obtained.

The same strategy was used to determine the human sequence as discussedabove for the mouse sequence. An oligo (#30152,GACCTGGGCCTGGGCTACGCGTCCGACGAG, SEQ ID NO:62) was used as a probe in aSouthern blot analysis to identify restriction fragments of the P1Clones which contained the human neurturin coding sequence. Theserestriction fragments (Eag I, Pvu II, Hind III, Kpn I) were subclonedinto the Bluescript KS plasmid and sequenced.

The results of subcloning and sequencing of human genomic fragments wereas follows. The Eag I fragment was found to be approximately 6 kb insize with the 3' Eag I site located 60 bp downstream from the stopcodon. The Pvu II fragment was approximately 3.5 kb in size with the 3'Pvu II site located 250 bp downstream from the stop codon. The Hind IIIfragment was approximately 4.8 kb in size with the 3' Hind III sitelocated 3kb downstream from the stop codon. The Kpn I fragment wasapproximately 4.2 kb in size with the 3' Kpn I site located 3.1 kbdownstream from the stop codon.

The second coding exon was sequenced using these subcloned fragments. Inaddition, sequence was obtained from 250 bp flanking the 3' side of thesecond exon. The sequence was also obtained from 1000 bp flanking the 5'side of the coding exon. From these flanking sequences, forward primer30341 (5'-CTGGCGTCCCAMCAAGGGTCTTCG-3', SEQ ID NO:71) and reverse primer30331 (5'-GCCAGTGGTGCCGTCGAGGCGGG-3', SEQ ID NO:72) were designed sothat the entire coding sequence of the second exon could be amplified byPCR.

The first coding exon was not mapped relative to the restriction sitesabove but was contained in the Eag I fragment. The sequence of this exonwas obtained from the subcloned Eag I fragment using the mouse primer466215 (5'-GGCCCAGGATGAGGCGCTGGAAGG-3', SEQ ID NO:73), which containsthe ATG initiation codon. Further sequence of the first coding exon wasobtained with reverse primer 20215 (5'-CCACTCCACTGCCTGAWATTCWACCCC-3',SEQ ID NO:74), designed from the sequence obtained with primer 466215.Forward primer 20205 (5'-CCATGTGATTATCGACCATTCGGC-3', SEQ ID NO:75) wasdesigned from sequence obtained with primer 20215. Primers 20205 and20215 flank the coding sequence of the first coding exon and can be usedto amplify this coding sequence using PCR.

EXAMPLE 6

This example illustrates the preparation of expression vectorscontaining neurturin cDNA.

For expression of recombinant neurturin in mammalian cells the neurturinvector pCMV-NTN-3-1 was constructed. The 585 nucleotide open readingframe of the neurturin cDNA was amplified by PCR using a primercontaining the first 27 nucleotides of the neurturin coding sequence (5'-GCGACGCGTACCATGAGGCGCTGGAAGGCAGCGGCCCTG, SEQ ID NO:63) and a primercontaining the last 5 codons and the stop codon(5'-GACGGATCCGCATCACACGCACGCGCACTC) (SEQ ID NO:64) using reversetranscribed postnatal day 1 mouse brain mRNA as template using (PCRparameters: 94° C. for 30 sec; 60° C. for 15 see; and 68° C. for 2 minfor 35 cycles and including 5% DMSO in the reaction). The PCR productwas subcloned into the Eco RV site of BSKS and sequenced to verify thatit contained no PCR generated mutations. The neurturin coding sequencewas then excised from this vector using Mlu I (5' end) and Bam H1 (3'end) and inserted downstream of the CMV IE promoter/enhancer in themammalian expression vector pCB6 (Brewer, C. B. Methods in Cell Biology43:233-245, 1994) to produce the pCMV-NTN-3-1 vector using these sites.

For expression of recombinant protein in E. Coli, the mature codingregion of mouse neurturin was amplified by PCR using a primer containingthe first 7 codons of the mature coding sequence(5'-GACCATATGCCGGGGGCTCGGCCTTGTGG) (SEQ ID NO:65) and a primercontaining the last 5 codons and the stop codon5'-GACGGATCCGCATCACACGCACGCGCACTC (SEQ ID NO:66) using a fragmentcontaining the murine neurturin gene as template using (PCR parameters:94° C. for 30 sec; 60° C. for 15 sec and 68° C. for 90 sec for 25 cycleswith 5%=DMSO added into the reaction). The amplified product wassubcloned into the Eco RV site of BSKS, the nucleotide sequence wasverified, and this fragment was then transferred to the expressionvector pET-30a (Novagen, Madison, Wis.) using an Nde 1 site (5' end) andan Eco RI site (3' end). The pET-neurturin (pET-NTN) vector codes for aninitiator methionine in front of the first amino acid of the maturemouse neurturin protein predicted from the N-terminal amino acidsequence of neurturin purified from the CHO cell conditioned media.

EXAMPLE 7

This example illustrates the transient transfection of NIH3T3 cells withthe neurturin expression vector pCMV-NTN-3-1 and that the product of thegenomic sequence in Example 5 is biologically active.

To demonstrate that the cloned neurturin cDNA was sufficient to directthe synthesis of biologically active neurturin we transiently introducedthe pCMV-NTN-3-1 plasmid into NIH3T3 cells using the lipofectaminemethod of transfection. NIH3T3 cells were plated at a density of 400,000cells per well (34.6 mm diameter) in 6 well plates (Corning, Corning,N.Y.) 24 hours before transfection. DNA liposome complexes were preparedand added to the cells according to the manufacturer's protocol using1.5 μg CMV-neurturin plasmid DNA (isolated and purified using a Qiagen(Chatsworth, Calif.) tip-500 column according to manufacturer'sprotocol) and 10 μl lipofectamine reagent (Gibco BRL, Gaithersburg, Md.)in 1:1 DME/F12 medium containing 5 μg/ml insulin, 5 μg/ml transferrin,and 5 ng/ml sodium selenite (Sigma, St. Louis, Mo.). Five hours afterthe addition of DNA liposome complexes in 1 ml medium per well, 1 ml DMEmedium containing 20% calf serum was added to each well. Twenty-fourhours after the addition of DNA-liposome complexes, the 2 ml mediumabove was replaced with 1 ml DME medium containing 10% calf serum, 2 mMglutamine, 100 U/ml penicillin, 100 μ/ml streptomycin, and 25 ug/mlheparin. The cells were incubated for an additional 24 hours before theconditioned medium was harvested, centrifuged to remove cellular debris,and frozen.

As a control, NIH3T3 cells were transfected as above using 1.5 μgCMV-neo expression plasmid (containing no cDNA insert) in place of the1.5 μg CMV-neurturin plasmid. Conditioned medium from NIH3T3 cellstransfected with either control plasmid or CMV-neurturin plasmid wasassayed by direct addition to the SCG culture medium at the time of NGFdeprivation. Addition of 0.25 ml conditioned medium fromCMV-neurturin-transfected cells promoted 70% survival of sympatheticneurons, and >90% survival could be obtained with 0.45 ml of thisconditioned medium. No significant survival promoting activity wasdetected in the conditioned medium of control transfected NIH3T3 cells.

EXAMPLE 8

This example illustrates the preparation of Chinese hamster ovary cellsstably transformed with neurturin cDNA.

DG44 cells, a Chinese hamster ovary cell derivative that is deficient indihydrofolate reductase (DHFR) (Urlaub et al Cell 3:405-412, 1983 whichis incorporated by reference), were stably co-transfected withexpression plasmid (pCMV-NTN-3-1) and a DHFR expression plasmid (HLD)(McArthur, and Stanners J. Biol. Chem. 266:6000-6005, 1991 which isincorporated by reference).

On day 1 DG44 cells were plated at 1×10⁶ cells per 10 cm plate in Ham'sF12 medium with 10% fetal calf serum (FCS). This density must not beexceeded or cells will overgrow before selection media is added on day5.

On day 2 cells were transfected with a 9:1 ratio of pCMV-NTN to DHFRexpression plasmid using the calcium phosphate method (10 ug DNA /10 cmplate) (Chen and Okayama, Mol Cell Biol 7:2745-2752, 1987 which isincorporated by reference).

On day 3 the transfected cells were washed with Ham's F12 medium and fedHam's F12 with 10% FCS.

On day 5 the cells were washed with MEM alpha medium and fed selectionmedium, which is MEM alpha with 10% FCS and 400 ug/ml G418. The cellswere maintained in selection media, feeding every 4 days. Colonies beganto appear approximately 14 days after transfection. Colonies growing inselection media were then transferred to a 24 well plate and trypsinizedthe next day to disperse the cells. The cells were grown to confluencein either 24 well or 6 well plates in order to screen the cells forexpression of recombinant protein. Expression of neurturin was examinedin 10 clonal lines and two high expressing lines were detected using theSCG survival assay. These clonal lines were expanded and expression inthese selected cell lines was amplified by selection in 50 nMmethotrexate (MTX). For selection in MTX, cells were grown to 50%confluence in a 150 cm² flask in selection medium. The medium waschanged to MEM alpha containing 50 nM MTX concentration (it was notnecessary to use G418 during MTX amplification). After placement in 50nM MTX, the majority of cells died and colonies of resistant cellsreappeared in 1-2 weeks. At this time, the cells were trypsinized todisperse colonies and are split when cells reach confluence. Cellseventually reached the same growth rate as before. The selected cellswere screened for expression of recombinant protein. A 2-3 fold increasein expression was observed after selection in 50 nM MTX. Frozen stockswere kept for cell lines obtained from the original selection and the 50nM MTX selection. Further selection could be continued in increasing MTXuntil desired levels of expression are obtained.

Using the above method, we isolated cells identified asDG44CHO5-3(G418)(pCMV-NTN-3-1) and DG44CHO5-3(50nMMTX)(pCMV-NTN-3-1).Cells from the DG44CHO5-3(50nMMTX)(pCMV-NTN-3-1) strain expressed levelsof approximately 100 μg of biologically active protein per liter ofconditioned media determined by direct assay of conditioned medium inSCG assay according to the methods in example 1.

EXAMPLE 9

This example illustrates the expression of neurturin in various tissues.

A survey of neurturin and GDNF expression was performed in rat embryonictissues (E10, day 10 after conception), neonatal tissues (P1, PostnatalDay 1), and adult tissues (>3 mos) using semi-quantitative RT/PCR (Estuset al., J Cell Biol 127:1717-1727, 1994 which is incorporated byreference). The RNA samples were obtained from various tissues and PCRproducts were detected either by autoradiography after incorporation ofα-³² P-dCTP in the PCR and electrophoresis on a polyacrylamide gel (FIG.6) or by ethidium bromide staining of DNA after electrophoresis onagarose gels (Tables 3 and 4). The neurturin fragment of 101 base pairswas obtained using the forward primer CAGCGACGACGCGTGCGCAAAGAGCG (SEQ IDNO:67) and reverse primer TAGCGGCTGTGTACGTCCAGGAAGGACACCTCGT (SEQ IDNO:68) and the GDNF fragment of 194 base pairs was obtained using theforward primer AAAAATCGGGGGTGYGTCTTA (SEQ ID NO:69) and the reverseprimer CATGCCTGGCCTACYTTGTCA (SEQ ID NO:70).

No neurturin or GDNF mRNA was detected at the earliest embryonic age(embryonic day 10, E10) surveyed.

In neonates (postnatal day 1, P1) both transcripts were expressed inmany tissues although neurturin tended to show a greater expression inmost tissues than did GDNF. (see table 3).

                  TABLE 3                                                         ______________________________________                                                     NEURTURIN                                                                              GDNF                                                    ______________________________________                                        Liver          +++        -                                                   Blood          +++        +                                                   Thymus         +          -                                                   Brain          ++         +                                                   Sciatic        -          +                                                   nerve                                                                         Kidney         ++         ++                                                  Spleen         ++         +                                                   Cerebellum     ++         +                                                   Heart          ++         +                                                   Bone           +          +                                                   ______________________________________                                    

As shown in Table 3, differences in the tissue distributions ofneurturin and GDNF were noted. In particular, no GDNF was detected inliver and thymus where neurturin expression was detected and noneurturin was detected in sciatic nerve where GDNF was detected.

Neurturin and GDNF mRNA were detected in many tissues in the adultanimal, but the tissue-specific pattern of expression for these twogenes was very different. (table 4, FIG. 5).

                  TABLE 4                                                         ______________________________________                                                     NEURTURIN                                                                              GDNF                                                    ______________________________________                                        Liver          -          -                                                   Blood          +          -                                                   Thymus         +          ++                                                  Brain          +          -                                                   Sciatic        -          -                                                   nerve                                                                         Kidney         ++         +                                                   Spleen         -          +                                                   Cerebellum     -          -                                                   Uterus         ++         -                                                   Bone marrow    ++         -                                                   Testis         ++         ++                                                  Ovary          +          +                                                   Placenta       +          -                                                   Skeletal       +          -                                                   muscle                                                                        Spinal cord    +          -                                                   Adrenal        ++         ++                                                  gland                                                                         Gut            +          ++                                                  ______________________________________                                    

As shown in table 4, neurturin was found to be expressed in brain andspinal cord as well as in blood and bone marrow where no GDNF wasdetected. The level of expression of neurturin in brain and blood was,however, less than that detected in neonatal tissue.

Neurturin was also highly expressed in freshly isolated rat peritonealmast cells, whereas GDNF showed little or no expression.

EXAMPLE 10

This example illustrates the preparation of antisera to neurturin byimmunization of rabbits with a neurturin peptide.

The peptide sequence corresponding to amino acids 73-87 of the maturemurine neurturin protein was synthesized and coupled to keyhole limpethemocyanin (KLH) as described earlier (Harlow and Lane, Antibodies: alaboratory manual, 1988. Cold Spring Harbor Laboratory, New York, N.Y.p. 72-81 which is incorporated by reference). The KLH-coupled peptidewas submitted to Caltag, Inc. and each of two rabbits were immunized.Immunization was by subcutaneous injection at 7-10 sites. The firstinjection was with 150 μg KLH-coupled peptide which was resuspended in0.5 ml saline and emulsified with 0.5 ml complete Freund's adjuvant.Boost injections were begun 4 weeks after the initial injection and wereperformed once every 7 days as above for a total of 5 injections exceptthat 100 μg of KLH-coupled peptide and incomplete Freund's adjuvant wereused. Serum samples were collected 1 week after the fifth boost.

A pooled volume of twenty ml of serum that had been collected from bothrabbits one week after the 5th injection was purified. For purification,a peptide affinity column was prepared by coupling the above peptide tocyanogen bromide activated Sepharose 4B according to the manufacturersprotocol (Pharmacia Biotech). The serum was diluted 10 fold in 10 mMTris pH 7.5 buffer and mixed by gentle rocking for 16 hours at 4° C.with 0.5 ml of peptide agarose matrix containing 5 mg of coupledpeptide. The matrix was placed into a column, washed with 5 ml of 10 mMTris pH 7.5, 150 mM NaCl, washed with 5 ml of 10 mM Tris pH 7.5 buffercontaining 0.4M NaCl and eluted with 5.5 ml of 100 mM glycine pH 2.5buffer. One tenth volume of 1.0M Tris pH 8.0 buffer was added to theeluate immediately after elution to neutralize the pH. The glycineeluate was dialyzed overnight against 10 mM Tris pH 7.5, 150 mM NaCl.

The affinity-purified antibodies were used in a western blot todemonstrate specific recognition of recombinant neurturin protein. Tenml of conditioned medium collected from DG44CHO5-3(G418)(pCMV-NTN-3-1)cells was purified over SP Sepharose as described in Example 1 and theproteins electrophoresed on a reducing SDS-PAGE gel in the tricinebuffer system (Schagger and von Jagow Analytical Biochemistry166:368-379, 1987). The proteins were electoblotted to a nitrocellulosemembrane in 25 mM Tris, 192 mM glycine, 0.04% SDS, 17% methanol at 4° C.for 16 hr. The membrane was incubated with the affinity-purifiedanti-neurturin peptide antibodies and then with horseradishperoxidase-coupled sheep anti-rabbit IgG (Harlow and Lane, supra, p.498-510). Bound antibodies were detected with enhanced chemiluminescence(ECL kit, Amersham, Buckinghamshire, England). The anti-neurturinantibodies recognized a single, approximately 11.5 kD protein band inthe conditioned medium of the DG44CHO5-3(G418)(pCMV-NTN-3-1) cells.Using these anti-neurturin antibodies, neurturin protein could bedetected in 10 ml of conditioned medium fromDG44CHO5-3(G418)(pCMV-NTN-3-1) cells but could not be detected in 10 mlof medium conditioned with DG44 cells that had not been transformed withthe neurturin expression vector.

EXAMPLE 11

The following example illustrates the identification of additionalmembers of the GDNF/neurturin gene subfamily.

The TGF-β superfamily currently contains over 25 different gene members(for review see Kingsley, Genes and Development 8: 133-146, 1994 whichis incorporated by reference). The individual family members displayvarying degrees of homology with each other and several subgroups withinthe superfamily can be defined by phylogenetic analysis using theClustal V program (Higgins et al, Comput Appl Biosct 8: 189-191, 1992which is incorporated by reference) and by bootstrap analysis ofphylogenetic trees (Felsenstein, Evolution 39:783-791, 1985 which isincorporated by reference). Neurturin is approximately 40% identical toGDNF but less than 20% identical to any other member of the TGF-βsuperfamily. Several sequence regions in neurturin can be identified(FIG. 5) that are highly conserved within the GDNF/neurturin subfamilybut not within the TGF-β superfamily. These conserved regions are likelyto characterize a subfamily containing previously unisolated genes,which can now be isolated using the conserved sequence regionsidentified by the discovery and sequencing of the neurturin gene.Regions of high sequence conservation between neurturin and GDNF allowthe design of degenerate oligonucleotides which can be used either asprobes or primers. Conserved-region amino acid sequences have beenidentified herein to include Val-Xaa₁ -Xaa₂ -Leu-Gly-Leu-Gly-Tyr inwhich Xaa₁ is Ser or Thr and Xaa₂ is Glu or Asp (SEQ ID NO:33); Glu-Xaa₁-Xaa₂ -Xaa₃ -Phe-Arg-Tyr-Cys-Xaa₄ -Gly-Xaa₅ -Cys-Xaa₆ -Xaa₇ -Ala inwhich Xaa₁ is Thr or Glu, Xaa₂ is Val or Leu, Xaa3 is Leu or Ile, Xaa₄is Ala or Ser, Xaa₅ is Ala or Ser, Xaa₆ is Glu or Asp and Xaa₇ is Ala orSer (SEQ ID NO:34); and Cys-Cys-Arg-Pro-Xaa₁ -Ala-Xaa₂ -Xaa₃ -Asp-Xaa₄-Xaa₅ -Ser-Phe-Leu-Asp in which Xaa₁ is Thr or Val or Ile, Xaa2 is Tyror Phe, Xaa₃ is Glu or Asp, Xaa₄ is Glu or Asp and Xaa₅ is val or leu(SEQ ID NO:35). Nucleotide sequences containing a coding sequence forthe above conserved sequences or fragments of the above conservedsequences can be used as probes. Exemplary probe and primer sequenceswhich can be designed from these regions are Primer 1,GTNWSNGANYTNGGNYTNGGNTA (SEQ ID NO:42) which encodes the amino acidsequence, Val-Xaa₁ -Xaa₂ -Leu-Gly-Leu-Gly-Tyr where Xaa₁ is Ser or Thrand Xaa₂ is Glu or Asp (SEQ ID NO:33); Primer 2,TTYMGNTAYTGYDSNGGNDSNTGYGANKCNGC (SEQ ID NO:43) which encodes amino acidsequence Phe-Arg-Tyr-Cys-Xaa₁ -Gly-Xaa₂ -Cys-Xaa₃ -Xaa₄ -Ala where Xaa₁is Ala or Ser, Xaa₂ is Ala or Ser, Xaa₃ is Glu or Asp and Xaa₄ is Ser orAla (SEQ ID NO:36); Primer 3 reverse GCNGMNTCRCANSHNCCNSHRTANCKRAA (SEQID NO:44) which encodes amino acid sequence Phe-Arg-Tyr-Cys-Xaa-Gly-Xaa₂-Cys-Xaa₃ -Xaa₄ -Ala where Xaa₁ is Ala or Ser, Xaa₂ is Ala or Ser, Xaa3is Glu or Asp and Xaa₄ is Ser or Ala (SEQ ID NO:37); Primer 4 reverseTCRTCNTCRWANGCNRYNGGNCKCARCA (SEQ ID NO:45) which encodes amino acidsequence amino acid sequence Cys-Cys-Arg-Pro-Xaa₁ -Ala-Xaa₂ -Xaa₃-Asp-Xaa₄ where Xaa₁ is Ile or Thr or Val, Xaa₂ Try or Phe, Xaa₃ is Gluor Asp and Xaa₄ is Glu or Asp (SEQ ID NO:38); Primer 5 reverseTCNARRAANSWNAVNTCRTCNTCRWANGC (SEQ ID NO:46) which encodes amino acidsequence Ala-Xaa₁ -Xaa₂ -Asp-Xaa₃ -Xaa₄ -Ser-Phe-Leu-Asp where Xaa₁ isTyr or Phe, Xaa₂ Glu or Asp, Xaa₃ is Glu or Asp, and Xaa₄ is Val or Leu(SEQ ID NO:39); Primer 6 GARRMNBTNHTNTTYMGNTAYTG (SEQ ID NO:47) whichencodes amino acid sequence Glu-Xaa₁ -Xaa₂ -Xaa₃ -Phe-Arg-Tyr-Cys whereXaa₁ is Glu or Thr, Xaa₂ is Leu or Val and Xaa₃ is Ile or Leu (SEQ IDNO:40); Primer 7 GARRMNBTNHTNTTYMGNTAYTGYDSNGGNDSNTGHGA (SEQ ID NO:48)which encodes amino acid sequence Glu-Xaa₁ -Xaa₂ -Xaa₃-Phe-Arg-Tyr-Cys-Xaa₄ -Gly-Xaa₅ -Cys-Xaa₆ where Xaa₁ is Glu or Thr, Xaa₂is Leu or Val, Xaa₃ is lie or Leu, Xaa₄ is Ser or Ala, Xaa₅ is Ser orAla and Xaa₆ is Glu or Asp (SEQ ID NO:41).

The above sequences can be used as probes for screening libraries ofgenomic clones or as primers for amplifying gene fragments from genomicDNA or libraries of genomic clones or from reverse transcribed cDNAusing RNA templates from a variety of tissues. Genomic DNA or librariesof genomic clones can be used as templates because the intron/exonstructures of neurturin and GDNF are conserved and coding sequences ofthe mature proteins are not interrupted by intron.

A degenerate oligonucleotide can be synthesized as a mixture ofoligonucleotides containing all of the possible nucleotide sequenceswhich code for the conserved amino acid sequence. To reduce the numberof different oligonucleotides in a degenerate mix, an inosine base canbe incorporated in the synthesis at positions where all four nucleotidesare possible. The inosine base forms base pairs with each of the fournormal DNA bases which are less stabilizing than AT and GC base pairsbut which are also less destabilizing than mismatches between the normalbases (i.e. AG, AC, TG, TC).

To isolate family members a primer above can be end labeled with ³² Pusing T4 polynucleotide kinase and hybridized to libraries of humangenomic clones according to standard procedures.

A preferred method for isolating family member genes would be to usevarious combinations of the degenerate primers above as primers in thepolymerase chain reaction using genomic DNA as a template. As an exampleprimer 2 (SEQ ID NO:43) can be used with primer 4 (SEQ ID NO:45) in PCRwith 1 ug of human genomic DNA and cycling parameters of 94° C. for 30sec, 50° C. for 30 sec, and 72° C. for 60 sec. These PCR conditions areexemplary only and one skilled in the art will readily appreciate that arange of suitable conditions could be used or optimized such asdifferent temperatures and varying salt concentrations in the buffermedium and the like. It is preferred that DMSO be added to the PCRreaction to a final concentration of 5% inasmuch as this was found to benecessary for amplification of this region of the neurturin gene. ThePCR reaction, when run on an agarose gel, should contain products in thesize range of 125-150 base pairs since a one amino acid gap isintroduced in the neurturin sequence when aligned with GDNF, and thusfamily member genes might also contain a slightly variable spacingbetween the conserved sequences of primers 2 and 4. The PCR products inthe range of 125-150 base pairs should contain multiple amplified geneproducts including GDNF and neurturin as well as previously unisolatedfamily members. To identify sequences of these products, they can be gelpurified and ligated into the Bluescript plasmid (Stratagene), and thentransformed into the XL1-blue E. Coli host strain (Stratagene).Bacterial colonies containing individual subcloned can be picked forisolation and plated on nitrocellulose filters in two replicas. Each ofthe replicate filters can be screened with an oligonucleotide probe foreither unique GDNF or unique neurturin sequence in the amplified region.Subcloned not hybridizing to either GDNF or neurturin can be sequencedand if found to encode previously unisolated family members, thesequence can be used to isolate full length cDNA clones and genomicclones as was done for neurturin (Example 5). A similar method was usedto isolate new gene members (GDF-3 and GDF-9) of the TGF-β superfamilybased on homology between previously identified genes (McPherron J BiolChem 268: 3444-3449, 1993 which is incorporated by reference).

The inventors herein believe that the most preferred way to isolatefamily member genes may be to apply the above PCR procedure as ascreening method to isolate individual family member genomic clones froma library. This is because there is only one exon for the coding regionof both mature neurturin and GDNF. If, for example, the above PCRreaction with primers 2 and 4 generates products of the appropriate sizeusing human genomic DNA as template, the same reaction can be performedusing, as template, pools of genomic clones in the P1 vector accordingto methods well known in the art, for example that used for isolatingneurturin human genomic clones (Example 5). Pools containing theneurturin gene in this library have previously been identified and GDNFcontaining pools can be readily identified by screening with GDNFspecific primers. Thus non-neurturin, non-GDNF pools which generate aproduct of the correct size using the degenerate primers will be readilyrecognized as previously unisolated family members. The PCR productsgenerated from these pools can be sequenced directly using the automatedsequencer and genomic clones can be isolated by further subdivision andscreening of the pooled clones as a standard service offered by GenomeSystems, Inc.

Deposit of Strain, the following strain is on deposit under the terms ofthe Budapest Treaty, with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. The accession number indicated wasassigned after successful viability testing, and the requisite fees werepaid. Access to said cultures will be available during pendency of thepatent application to one determined by the Commissioner to be entitledthereto under 37 CFR 1.14 and 35 USC 122. All restriction onavailability of said cultures to the public will be irrevocably removedupon the granting of a patent based upon the application. Moreover, thedesignated deposits will be maintained for a period of thirty (30) yearsfrom the date of deposit, or for five (5) years after the last requestfor the deposit, or for the enforceable life of the U.S. patent,whichever is longer. Should a culture become nonviable or beinadvertently destroyed, or, in the case of plasmid-containing strains,lose its plasmid, it will be replaced with a viable culture. Thedeposited materials mentioned herein are intended for convenience only,and are not required to practice the present invention in view of thedescription herein, and in addition, these materials are incorporatedherein by reference.

    ______________________________________                                        Strain           Deposit Date ATCC No.                                        DG44CHO-PHSP-NGFI-B                                                                            August 25, 1995                                              ______________________________________                                    

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 78                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaArgLeuGlyAlaArgProCysGlyLeuArgGluLeuGluValArg                              151015                                                                        ValSerGluLeuGlyLeuGlyTyrAlaSerAspGluThrValLeuPhe                              202530                                                                        ArgTyrCysAlaGlyAlaCysGluAlaAlaAlaArgValTyrAspLeu                              354045                                                                        GlyLeuArgArgLeuArgGlnArgArgArgLeuArgArgGluArgVal                              505560                                                                        ArgAlaGlnProCysCysArgProThrAlaTyrGluAspGluValSer                              65707580                                                                      PheLeuAspAlaHisSerArgTyrHisThrValHisGluLeuSerAla                              859095                                                                        ArgGluCysAlaCysVal                                                            100                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProGlyAlaArgProCysGlyLeuArgGluLeuGluValArgValSer                              151015                                                                        GluLeuGlyLeuGlyTyrThrSerAspGluThrValLeuPheArgTyr                              202530                                                                        CysAlaGlyAlaCysGluAlaAlaIleArgIleTyrAspLeuGlyLeu                              354045                                                                        ArgArgLeuArgGlnArgArgArgValArgArgGluArgAlaArgAla                              505560                                                                        HisProCysCysArgProThrAlaTyrGluAspGluValSerPheLeu                              65707580                                                                      AspValHisSerArgTyrHisThrLeuGlnGluLeuSerAlaArgGlu                              859095                                                                        CysAlaCysVal                                                                  100                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "ANY AMINO ACID"                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerGlyAlaArgProXaaGlyLeuArgGluLeuGluValSerValSer                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "ANY AMINO ACID"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "SERINE OR CYSTEINE"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       XaaCysAlaGlyAlaXaaGluAlaAlaVal                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "ANY AMINO ACID"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "ANY AMINO ACID"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note= "GLUTAMINE OR GLUTAMIC ACID"                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       XaaXaaValGluAlaLysProCysCysGlyProThrAlaTyrGluAsp                              151015                                                                        XaaValSerPheLeuSerVal                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrHisThrLeuGlnGluLeuSerAlaArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 197 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetGlnArgTrpLysAlaAlaAlaLeuAlaSerValLeuCysSerSer                              151015                                                                        ValLeuSerIleTrpMetCysArgGluGlyLeuLeuLeuSerHisArg                              202530                                                                        LeuGlyProAlaLeuValProLeuHisArgLeuProArgThrLeuAsp                              354045                                                                        AlaArgIleAlaArgLeuAlaGlnTyrArgAlaLeuLeuGlnGlyAla                              505560                                                                        ProAspAlaMetGluLeuArgGluLeuThrProTrpAlaGlyArgPro                              65707580                                                                      ProGlyProArgArgArgAlaGlyProArgArgArgArgAlaArgAla                              859095                                                                        ArgLeuGlyAlaArgProCysGlyLeuArgGluLeuGluValArgVal                              100105110                                                                     SerGluLeuGlyLeuGlyTyrAlaSerAspGluThrValLeuPheArg                              115120125                                                                     TyrCysAlaGlyAlaCysGluAlaAlaAlaArgValTyrAspLeuGly                              130135140                                                                     LeuArgArgLeuArgGlnArgArgArgLeuArgArgGluArgValArg                              145150155160                                                                  AlaGlnProCysCysArgProThrAlaTyrGluAspGluValSerPhe                              165170175                                                                     LeuAspAlaHisSerArgTyrHisThrValHisGluLeuSerAlaArg                              180185190                                                                     GluCysAlaCysVal                                                               195                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetArgArgTrpLysAlaAlaAlaLeuValSerLeuIleCysSerSer                              151015                                                                        LeuLeuSerValTrpMetCysGlnGluGlyLeuLeuLeuGlyHisArg                              202530                                                                        LeuGlyProAlaLeuAlaProLeuArgArgProProArgThrLeuAsp                              354045                                                                        AlaArgIleAlaArgLeuAlaGlnTyrArgAlaLeuLeuGlnGlyAla                              505560                                                                        ProAspAlaValGluLeuArgGluLeuSerProTrpAlaAlaArgIle                              65707580                                                                      ProGlyProArgArgArgAlaGlyProArgArgArgArgAlaArgPro                              859095                                                                        GlyAlaArgProCysGlyLeuArgGluLeuGluValArgValSerGlu                              100105110                                                                     LeuGlyLeuGlyTyrThrSerAspGluThrValLeuPheArgTyrCys                              115120125                                                                     AlaGlyAlaCysGluAlaAlaIleArgIleTyrAspLeuGlyLeuArg                              130135140                                                                     ArgLeuArgGlnArgArgArgValArgArgGluArgAlaArgAlaHis                              145150155160                                                                  ProCysCysArgProThrAlaTyrGluAspGluValSerPheLeuAsp                              165170175                                                                     ValHisSerArgTyrHisThrLeuGlnGluLeuSerAlaArgGluCys                              180185190                                                                     AlaCysVal                                                                     195                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 306 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCGCGGTTGGGGGCGCGGCCTTGCGGGCTGCGCGAGCTGGAGGTGCGCGTGAGCGAGCTG60                GGCCTGGGCTACGCGTCCGACGAGACGGTGCTGTTCCGCTACTGCGCAGGCGCCTGCGAG120               GCTGCCGCGCGCGTCTACGACCTCGGGCTGCGACGACTGCGCCAGCGGCGGCGCCTGCGG180               CGGGAGCGGGTGCGCGCGCAGCCCTGCTGCCGCCCGACGGCCTACGAGGACGAGGTGTCC240               TTCCTGGACGCGCACAGCCGCTACCACACGGTGCACGAGCTGTCGGCGCGCGAGTGCGCC300               TGCGTG306                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 300 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCGGGGGCTCGGCCTTGTGGGCTGCGCGAGCTCGAGGTGCGCGTGAGCGAGCTGGGCCTG60                GGCTACACGTCGGATGAGACCGTGCTGTTCCGCTACTGCGCAGGCGCGTGCGAGGCGGCC120               ATCCGCATCTACGACCTGGGCCTTCGGCGCCTGCGCCAGCGGAGGCGCGTGCGCAGAGAG180               CGGGCGCGGGCGCACCCGTGTTGTCGCCCGACGGCCTATGAGGACGAGGTGTCCTTCCTG240               GACGTGCACAGCCGCTACCACACGCTGCAAGAGCTGTCGGCGCGGGAGTGCGCGTGCGTG300               (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 591 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ATGCAGCGCTGGAAGGCGGCGGCCTTGGCCTCAGTGCTCTGCAGCTCCGTGCTGTCCATC60                TGGATGTGTCGAGAGGGCCTGCTTCTCAGCCACCGCCTCGGACCTGCGCTGGTCCCCCTG120               CACCGCCTGCCTCGAACCCTGGACGCCCGGATTGCCCGCCTGGCCCAGTACCGTGCACTC180               CTGCAGGGGGCCCCGGATGCGATGGAGCTGCGCGAGCTGACGCCCTGGGCTGGGCGGCCC240               CCAGGTCCGCGCCGTCGGGCGGGGCCCCGGCGGCGGCGCGCGCGTGCGCGGTTGGGGGCG300               CGGCCTTGCGGGCTGCGCGAGCTGGAGGTGCGCGTGAGCGAGCTGGGCCTGGGCTACGCG360               TCCGACGAGACGGTGCTGTTCCGCTACTGCGCAGGCGCCTGCGAGGCTGCCGCGCGCGTC420               TACGACCTCGGGCTGCGACGACTGCGCCAGCGGCGGCGCCTGCGGCGGGAGCGGGTGCGC480               GCGCAGCCCTGCTGCCGCCCGACGGCCTACGAGGACGAGGTGTCCTTCCTGGACGCGCAC540               AGCCGCTACCACACGGTGCACGAGCTGTCGGCGCGCGAGTGCGCCTGCGTG591                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 585 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ATGAGGCGCTGGAAGGCAGCGGCCCTGGTGTCGCTCATCTGCAGCTCCCTGCTATCTGTC60                TGGATGTGCCAGGAGGGTCTGCTCTTGGGCCACCGCCTGGGACCCGCGCTTGCCCCGCTA120               CGACGCCCTCCACGCACCCTGGACGCCCGCATCGCCCGCCTGGCCCAGTATCGCGCTCTG180               CTCCAGGGCGCCCCCGACGCGGTGGAGCTTCGAGAACTTTCTCCCTGGGCTGCCCGCATC240               CCGGGACCGCGCCGTCGAGCGGGTCCCCGGCGTCGGCGGGCGCGGCCGGGGGCTCGGCCT300               TGTGGGCTGCGCGAGCTCGAGGTGCGCGTGAGCGAGCTGGGCCTGGGCTACACGTCGGAT360               GAGACCGTGCTGTTCCGCTACTGCGCAGGCGCGTGCGAGGCGGCCATCCGCATCTACGAC420               CTGGGCCTTCGGCGCCTGCGCCAGCGGAGGCGCGTGCGCAGAGAGCGGGCGCGGGCGCAC480               CCGTGTTGTCGCCCGACGGCCTATGAGGACGAGGTGTCCTTCCTGGACGTGCACAGCCGC540               TACCACACGCTGCAAGAGCTGTCGGCGCGGGAGTGCGCGTGCGTG585                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGAGGGAGAGCGCGCGGTGGTTTCGTCCGTGTGCCCCGCGCCCGGCGCTCCTCGCGTGGC60                CCCGCGTCCTGAGCGCGCTCCAGCCTCCCACGCGCGCCACCCCGGGGTTCACTGAGCCCG120               GCGAGCCCGGGGAAGACAGAGAAAGAGAGGCCAGGGGGGGAACCCCATGGCCCGGCCCGT180               GTCCCGCACCCTGTGCGGTGGCCTCCTCCGGCACGGGGTCCCCGGGTCGCCTCCGGTCCC240               CGCGATCCGGATGGCGCACGCAGTGGCTGGGGCCGGGCCGGGCTCGGGTGGTCGGAGGAG300               TCACCACTGACCGGGTCATCTGGAGCCCGTGGCAGGCCGAGGCCCAGG348                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TGCTACCTCACGCCCCCCGACCTGCGAAAGGGCCCTCCCTGCCGACCCTCGCTGAGAACT60                GACTTCACATAAAGTGTGGGAACTCCC87                                                 (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetGlnArgTrpLysAlaAlaAlaLeuAlaSerValLeuCysSerSer                              151015                                                                        ValLeuSer                                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetArgArgTrpLysAlaAlaAlaLeuValSerLeuIleCysSerSer                              151015                                                                        LeuLeuSer                                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ATGCAGCGCTGGAAGGCGGCGGCCTTGGCCTCAGTGCTCTGCAGCTCCGTGCTGTCC57                   (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ATGAGGCGCTGGAAGGCAGCGGCCCTGGTGTCGCTCATCTGCAGCTCCCTGCTATCT57                   (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      IleTrpMetCysArgGluGlyLeuLeuLeuSerHisArgLeuGlyPro                              151015                                                                        AlaLeuValProLeuHisArgLeuProArgThrLeuAspAlaArgIle                              202530                                                                        AlaArgLeuAlaGlnTyrArgAlaLeuLeuGlnGlyAlaProAspAla                              354045                                                                        MetGluLeuArgGluLeuThrProTrpAlaGlyArgProProGlyPro                              505560                                                                        ArgArgArgAlaGlyProArgArgArgArgAlaArg                                          657075                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 228 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ATCTGGATGTGTCGAGAGGGCCTGCTTCTCAGCCACCGCCTCGGACCTGCGCTGGTCCCC60                CTGCACCGCCTGCCTCGAACCCTGGACGCCCGGATTGCCCGCCTGGCCCAGTACCGTGCA120               CTCCTGCAGGGGGCCCCGGATGCGATGGAGCTGCGCGAGCTGACGCCCTGGGCTGGGCGG180               CCCCCAGGTCCGCGCCGTCGGGCGGGGCCCCGGCGGCGGCGCGCGCGT228                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 228 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GTCTGGATGTGCCAGGAGGGTCTGCTCTTGGGCCACCGCCTGGGACCCGCGCTTGCCCCG60                CTACGACGCCCTCCACGCACCCTGGACGCCCGCATCGCCCGCCTGGCCCAGTATCGCGCT120               CTGCTCCAGGGCGCCCCCGACGCGGTGGAGCTTCGAGAACTTTCTCCCTGGGCTGCCCGC180               ATCCCGGGACCGCGCCGTCGAGCGGGTCCCCGGCGTCGGCGGGCGCGG228                           (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ValTrpMetCysGlnGluGlyLeuLeuLeuGlyHisArgLeuGlyPro                              151015                                                                        AlaLeuAlaProLeuArgArgProProArgThrLeuAspAlaArgIle                              202530                                                                        AlaArgLeuAlaGlnTyrArgAlaLeuLeuGlnGlyAlaProAspAla                              354045                                                                        ValGluLeuArgGluLeuSerProTrpAlaAlaArgIleProGlyPro                              505560                                                                        ArgArgArgAlaGlyProArgArgArgArgAlaArg                                          657075                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      MetGlnArgTrpLysAlaAlaAlaLeuAlaSerValLeuCysSerSer                              151015                                                                        ValLeuSerIleTrpMetCysArgGluGlyLeuLeuLeuSerHisArg                              202530                                                                        LeuGlyProAlaLeuValProLeuHisArgLeuProArgThrLeuAsp                              354045                                                                        AlaArgIleAlaArgLeuAlaGlnTyrArgAlaLeuLeuGlnGlyAla                              505560                                                                        ProAspAlaMetGluLeuArgGluLeuThrProTrpAlaGlyArgPro                              65707580                                                                      ProGlyProArgArgArgAlaGlyProArgArgArgArgAlaArg                                 859095                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      MetArgArgTrpLysAlaAlaAlaLeuValSerLeuIleCysSerSer                              151015                                                                        LeuLeuSerValTrpMetCysGlnGluGlyLeuLeuLeuGlyHisArg                              202530                                                                        LeuGlyProAlaLeuAlaProLeuArgArgProProArgThrLeuAsp                              354045                                                                        AlaArgIleAlaArgLeuAlaGlnTyrArgAlaLeuLeuGlnGlyAla                              505560                                                                        ProAspAlaValGluLeuArgGluLeuSerProTrpAlaAlaArgIle                              65707580                                                                      ProGlyProArgArgArgAlaGlyProArgArgArgArgAlaArg                                 859095                                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 285 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      ATGCAGCGCTGGAAGGCGGCGGCCTTGGCCTCAGTGCTCTGCAGCTCCGTGCTGTCCATC60                TGGATGTGTCGAGAGGGCCTGCTTCTCAGCCACCGCCTCGGACCTGCGCTGGTCCCCCTG120               CACCGCCTGCCTCGAACCCTGGACGCCCGGATTGCCCGCCTGGCCCAGTACCGTGCACTC180               CTGCAGGGGGCCCCGGATGCGATGGAGCTGCGCGAGCTGACGCCCTGGGCTGGGCGGCCC240               CCAGGTCCGCGCCGTCGGGCGGGGCCCCGGCGGCGGCGCGCGCGT285                              (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 285 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ATGAGGCGCTGGAAGGCAGCGGCCCTGGTGTCGCTCATCTGCAGCTCCCTGCTATCTGTC60                TGGATGTGCCAGGAGGGTCTGCTCTTGGGCCACCGCCTGGGACCCGCGCTTGCCCCGCTA120               CGACGCCCTCCACGCACCCTGGACGCCCGCATCGCCCGCCTGGCCCAGTATCGCGCTCTG180               CTCCAGGGCGCCCCCGACGCGGTGGAGCTTCGAGAACTTTCTCCCTGGGCTGCCCGCATC240               CCGGGACCGCGCCGTCGAGCGGGTCCCCGGCGTCGGCGGGCGCGG285                              (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 169 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      ATGCAGCGCTGGAAGGCGGCGGCCTTGGCCTCAGTGCTCTGCAGCTCCGTGCTGTCCATC60                TGGATGTGTCGAGAGGGCCTGCTTCTCAGCCACCGCCTCGGACCTGCGCTGGTCCCCCTG120               CACCGCCTGCCTCGAACCCTGGACGCCCGGATTGCCCGCCTGGCCCAGT169                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 425 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      ACCGTGCACTCCTGCAGGGGGCCCCGGATGCGATGGAGCTGCGCGAGCTGACGCCCTGGG60                CTGGGCGGCCCCCAGGTCCGCGCCGTCGGGCGGGGCCCCGGCGGCGGCGCGCGCGTGCGC120               GGTTGGGGGCGCGGCCTTGCGGGCTGCGCGAGCTGGAGGTGCGCGTGAGCGAGCTGGGCC180               TGGGCTACGCGTCCGACGAGACGGTGCTGTTCCGCTACTGCGCAGGCGCCTGCGAGGCTG240               CCGCGCGCGTCTACGACCTCGGGCTGCGACGACTGCGCCAGCGGCGGCGCCTGCGGCGGG300               AGCGGGTGCGCGCGCAGCCCTGCTGCCGCCCGACGGCCTACGAGGACGAGGTGTCCTTCC360               TGGACGCGCACAGCCGCTACCACACGGTGCACGAGCTGTCGGCGCGCGAGTGCGCCTGCG420               TGTGA425                                                                      (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 169 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ATGAGGCGCTGGAAGGCAGCGGCCCTGGTGTCGCTCATCTGCAGCTCCCTGCTATCTGTC60                TGGATGTGCCAGGAGGGTCTGCTCTTGGGCCACCGCCTGGGACCCGCGCTTGCCCCGCTA120               CGACGCCCTCCACGCACCCTGGACGCCCGCATCGCCCGCCTGGCCCAGT169                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      ATCGCGCTCTGCTCCAGGGCGCCCCCGACGCGGTGGAGCTTCGAGAACTTTCTCCCTGGG60                CTGCCCGCATCCCGGGACCGCGCCGTCGAGCGGGTCCCCGGCGTCGGCGGGCGCGGCCGG120               GGGCTCGGCCTTGTGGGCTGCGCGAGCTCGAGGTGCGCGTGAGCGAGCTGGGCCTGGGCT180               ACACGTCGGATGAGACCGTGCTGTTCCGCTACTGCGCAGGCGCGTGCGAGGCGGCCATCC240               GCATCTACGACCTGGGCCTTCGGCGCCTGCGCCAGCGGAGGCGCGTGCGCAGAGAGCGGG300               CGCGGGCGCACCCGTGTTGTCGCCCGACGGCCTATGAGGACGAGGTGTCCTTCCTGGACG360               TGCACAGCCGCTACCACACGCTGCAAGAGCTGTCGGCGCGGGAGTGCGCGTGCGTGTGA419                (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CysGlyLeuArgGluLeuGluValArgValSerGluLeuGlyLeuGly                              151015                                                                        TyrAlaSerAspGluThrValLeuPheArgTyrCysAlaGlyAlaCys                              202530                                                                        GluAlaAlaAlaArgValTyrAspLeuGlyLeuArgArgLeuArgGln                              354045                                                                        ArgArgArgLeuArgArgGluArgValArgAlaGlnProCysCysArg                              505560                                                                        ProThrAlaTyrGluAspGluValSerPheLeuAspAlaHisSerArg                              65707580                                                                      TyrHisThrValHisGluLeuSerAlaArgGluCysAlaCys                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CysGlyLeuArgGluLeuGluValArgValSerGluLeuGlyLeuGly                              151015                                                                        TyrThrSerAspGluThrValLeuPheArgTyrCysAlaGlyAlaCys                              202530                                                                        GluAlaAlaIleArgIleTyrAspLeuGlyLeuArgArgLeuArgGln                              354045                                                                        ArgArgArgValArgArgGluArgAlaArgAlaHisProCysCysArg                              505560                                                                        ProThrAlaTyrGluAspGluValSerPheLeuAspValHisSerArg                              65707580                                                                      TyrHisThrLeuGlnGluLeuSerAlaArgGluCysAlaCys                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "SERINE OR THREONINE"                           (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ValXaaXaaLeuGlyLeuGlyTyr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "THREONINE OR GLUTAMIC ACID"                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "VALINE OR LEUCINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "LEUCINE OR ISOLEUCINE"                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 13                                                              (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 14                                                              (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GluXaaXaaXaaPheArgTyrCysXaaGlyXaaCysXaaXaaAla                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "THREONINE OR VALINE OR                         ISOLEUCINE"                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "TYROSINE OR PHENYLALANINE"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "VALINE OR LEUCINE"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CysCysArgProXaaAlaXaaXaaAspXaaXaaSerPheLeuAsp                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "SERINE OR ALANINE"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      PheArgTyrCysXaaGlyXaaCysXaaXaaAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "ALANINE OR SERINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "SERINE OR ALANINE"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      PheArgTyrCysXaaGlyXaaCysXaaXaaAla                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "ISOLEUCINE OR THREONINE OR                     VALINE"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "TYROSINE OR PHENYLALANINE"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CysCysArgProXaaAlaXaaXaaAspXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "TYROSINE OR PHENYLALANINE"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "VALINE OR LEUCINE"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      AlaXaaXaaAspXaaXaaSerPheLeuAsp                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR THREONINE"                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "LEUCINE OR VALINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "ISOLEUCINE OR LEUCINE"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GluXaaXaaXaaPheArgTyrCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR THREONINE"                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "LEUCINE OR VALINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "ISOLEUCINE OR LEUCINE"                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "SERINE OR ALANINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /note= "SERINE OR ALANINE"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 13                                                              (D) OTHER INFORMATION: /note= "GLUTAMIC ACID OR ASPARTIC                      ACID"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GluXaaXaaXaaPheArgTyrCysXaaGlyXaaCysXaa                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GTNWSNGANYTNGGNYTNGGNTA23                                                     (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      TTYMGNTAYTGYDSNGGNDSNTGYGANKCNGC32                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GCNGMNTCRCANSHNCCNSHRCARTANCKRAA32                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TCRTCNTCRWANGCNRYNGGNCKRCARCA29                                               (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TCNARRAANSWNAVNTCRTCNTCRWANGC29                                               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GARRMNBTNHTNTTYMGNTAYTG23                                                     (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GARRMNBTNHTNTTYMGNTAYTGYDSNGGNDSNTGHGA38                                      (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      SerGlyAlaArgProXaaGlyLeuArgGluLeuGluValSerValSer                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      CCNACNGCNTAYGARGA17                                                           (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      AlaArgAlaHisProCysCysArgProThrAlaTyrGluAspGluVal                              151015                                                                        SerPheLeuAsp                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      ARYTCYTGNARNGTRTGRTA20                                                        (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      GACGAGGTGTCCTTCCTGGACGTACACA28                                                (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      TAGCGGCTGTGTACGTCCAGGAAGGACACCTCGT34                                          (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      CAGCGACGACGCGTGCGCAAAGAGCG26                                                  (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      TAYGARGACGAGGTGTCCTTCCTGGACGTACACAGCCGCTAYCAYAC47                             (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GCGGCCATCCGCATCTACGACCTGGG26                                                  (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CRTAGGCCGTCGGGCGRCARCACGGGT27                                                 (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GCGCCGAAGGCCCAGGTCGTAGATGCG27                                                 (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      CGCTACTGCGCAGGCGCGTGCGARGCGGC29                                               (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      CGCCGACAGCTCTTGCAGCGTRTGGTA27                                                 (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GAGCTGGGCCTGGGCTACGCGTCCGACGAG30                                              (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GCGACGCGTACCATGAGGCGCTGGAAGGCAGCGGCCCTG39                                     (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GACGGATCCGCATCACACGCACGCGCACTC30                                              (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GACCATATGCCGGGGGCTCGGCCTTGTGG29                                               (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      GACGGATCCGCATCACACGCACGCGCACTC30                                              (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      CAGCGACGACGCGTGCGCAAAGAGCG26                                                  (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      TAGCGGCTGTGTACGTCCAGGAAGGACACCTCGT34                                          (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      AAAAATCGGGGGTGYGTCTTA21                                                       (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      CATGCCTGGCCTACYTTGTCA21                                                       (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      CTGGCGTCCCAMCAAGGGTCTTCG24                                                    (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      GCCAGTGGTGCCGTCGAGGCGGG23                                                     (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      GGCCCAGGATGAGGCGCTGGAAGG24                                                    (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      CCACTCCACTGCCTGAWATTCWACCCC27                                                 (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      CCATGTGATTATCGACCATTCGGC24                                                    (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      SerProAspLysGlnMetAlaValLeuProArgArgGluArgAsnArg                              151015                                                                        GlnAlaAlaAlaAlaAsnProGluAsnSerArgGlyLysGlyArgArg                              202530                                                                        GlyGlnArgGlyLysAsnArgGlyCysValLeuThrAlaIleHisLeu                              354045                                                                        AsnValThrAspLeuGlyLeuGlyTyrGluThrLysGluGluLeuIle                              505560                                                                        PheArgTyrCysSerGlySerCysAspAlaAlaGluThrThrTyrAsp                              65707580                                                                      LysIleLeuLysAsnLeuSerArgAsnArgArgLeuValSerAspLys                              859095                                                                        ValGlyGlnAlaCysCysArgProIleAlaPheAspAspAspLeuSer                              100105110                                                                     PheLeuAspAspAsnLeuValTyrHisIleLeuArgLysHisSerAla                              115120125                                                                     LysArgCysGlyCysIle                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      SerProAspLysGlnAlaAlaAlaLeuProArgArgGluArgAsnArg                              151015                                                                        GlnAlaAlaAlaAlaSerProGluAsnSerArgGlyLysGlyArgArg                              202530                                                                        GlyGlnArgGlyLysAsnArgGlyCysValLeuThrAlaIleHisLeu                              354045                                                                        AsnValThrAspLeuGlyLeuGlyTyrGluThrLysGluGluLeuIle                              505560                                                                        PheArgTyrCysSerGlySerCysGluSerAlaGluThrMetTyrAsp                              65707580                                                                      LysIleLeuLysAsnLeuSerArgSerArgArgLeuThrSerAspLys                              859095                                                                        ValGlyGlnAlaCysCysArgProValAlaPheAspAspAspLeuSer                              100105110                                                                     PheLeuAspAspAsnLeuValTyrHisIleLeuArgLysHisSerAla                              115120125                                                                     LysArgCysGlyCysIle                                                            130                                                                           (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      SerProAspLysGlnAlaAlaAlaLeuProArgArgGluArgAsnArg                              151015                                                                        GlnAlaAlaAlaAlaSerProGluAsnSerArgGlyLysGlyArgArg                              202530                                                                        GlyGlnArgGlyLysAsnArgGlyCysValLeuThrAlaIleHisLeu                              354045                                                                        AsnValThrAspLeuGlyLeuGlyTyrGluThrLysGluGluLeuIle                              505560                                                                        PheArgTyrCysSerGlySerCysGluAlaAlaGluThrMetTyrAsp                              65707580                                                                      LysIleLeuLysAsnLeuSerArgSerArgArgLeuThrSerAspLys                              859095                                                                        ValGlyGlnAlaCysCysArgProValAlaPheAspAspAspLeuSer                              100105110                                                                     PheLeuAspAspSerLeuValTyrHisIleLeuArgLysHisSerAla                              115120125                                                                     LysArgCysGlyCysIle                                                            130                                                                           __________________________________________________________________________

What is claimed is:
 1. A method for providing trophic support forneurons in a patient in need thereof, the method comprisingadministering to the patient a polynucleotide encoding a neurturinpolypeptide comprising SEQ ID NO:31, SEQ ID NO:32 or conservativelysubstituted variants thereof.
 2. The method of claim 1, wherein theneurturin polypeptide comprises SEQ ID NO:1 or a conservativelysubstituted variant thereof.
 3. The method of claim 2, wherein thepolynucleotide comprises SEQ ID NO:9.
 4. The method of claim 1, whereinthe neurturin polypeptide comprises SEQ ID NO:7.
 5. The method of claim4, wherein the polynucleotide comprises SEQ ID NO:11.
 6. The method ofclaim 1, wherein the polynucleotide is administered by implanting cellstransformed with the polynucleotide into the patient, wherein said cellsexpress the neurturin polypeptide.
 7. The method of claim 6, wherein thecells are encapsulated in a semipermeable membrane.
 8. The method ofclaim 1, wherein the patient suffers from a condition selected from thegroup consisting of peripheral neuropathy, amyotrophic lateralsclerosis, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, ischemic stroke, acute brain injury, acute spinal chord injury,nervous system tumors, multiple sclerosis, and infection.
 9. The methodof claim 8, wherein the condition is peripheral neuropathy.
 10. Themethod of claim 8, wherein the condition is amyotrophic lateralsclerosis.
 11. The method of claim 8, wherein the condition isParkinson's disease.
 12. The method of claim 8, wherein the condition isAlzheimer's disease.
 13. The method of claim 8, wherein the condition ismultiple sclerosis.
 14. The method of claim 8, wherein the condition isacute brain injury.
 15. The method of claim 8, wherein the condition isacute spinal chord injury.